Double-stranded oligonucleotides

ABSTRACT

Antisense sequences, including duplex RNAi compositions, which possess improved properties over those taught in the prior art are disclosed. The invention provides optimized antisense oligomer compositions and method for making and using the both in in vitro systems and therapeutically. The invention also provides methods of making and using the improved antisense oligomer compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/062,380, filedApr. 3, 2008 which claims priority to U.S. Ser. No. 11/049,636, filed onFeb. 2, 2005, now abandoned, which claims the benefit of U.S.Provisional Application No. 60/615,408, filed on Sep. 30, 2004, and60/540,552, filed on Feb. 2, 2004 and is also a continuation-in-part ofU.S. Ser. No. 10/357,529, filed Feb. 3, 2003, now abandoned, and Ser.No. 10/357,826, filed Feb. 3, 2003, now abandoned, both of which claimthe benefit of U.S. Provisional Application No. 60/353,203, filed onFeb. 1, 2002, 60/436,238, filed Dec. 23, 2002, 60/438,608, filed Jan. 7,2003, and 60/353,381, filed Feb. 1, 2002. The entire contents of theaforementioned applications are hereby expressly incorporated herein byreference.

BACKGROUND OF THE INVENTION

Complementary oligonucleotide sequences are promising therapeutic agentsand useful research tools in elucidating gene function. However,oligonucleotide molecules of the prior art are often subject to nucleasedegradation when applied to biological systems. Therefore, it is oftendifficult to achieve efficient inhibition of gene expression (includingprotein synthesis) using such compositions.

In order to maximize the usefulness, such as the potential therapeuticactivity and in vitro utility, of oligonucleotides that arecomplementary to other sequences of interest, it would be of greatbenefit to improve upon the prior art oligonucleotides by designingimproved oligonucleotides having increased stability both against serumnucleases and cellular nucleases and nucleases found in other bodilyfluids.

SUMMARY OF THE INVENTION

The instant invention is based, at least in part, on the discovery thatdouble-stranded oligonucleotides comprising an antisense oligonucleotideand a protector oligonucleotide, are capable of inhibiting genefunction. Thus, the invention improves the prior art antisensesequences, inter alia, by providing oligonucleotides which are resistantto degradation by cellular nucleases.

Accordingly, the invention provides optimized oligonucleotidecompositions and methods for making and using both in in vitro, and invivo systems, e.g., therapeutically.

In one aspect, the invention pertains to a double-strandedoligonucleotide composition having the structure depicted in FIG. 1,where (1) N is a nucleomonomer in complementary oligonucleotide strandsof equal length and where the sequence of Ns corresponds to a targetgene sequence and (2) X and Y are each independently selected from agroup consisting of nothing; from about 1 to about 20 nucleotides of 5′overhang; from about 1 to about 20 nucleotides of 3′ overhang; and aloop structure consisting from about 4 to about 20 nucleomonomers, wherethe nucleomonomers are selected from the group consisting of G and A.The invention further includes compositions such as reaction mixturescomprising such double-stranded oligonucleotides, as well as methods forusing such double-stranded oligonucleotides.

An “overhang” is a relatively short single-stranded nucleotide sequenceon the 5′- or 3′-hydroxyl end of a double-stranded oligonucleotidemolecule (also referred to as an “extension,” “protruding end,” or“sticky end”).

In one embodiment, the number of Ns in each strand of the duplex isbetween about 12 and about 50. In other embodiments, the number of Ns ineach strand of the duplex is between about 12 and about 40 (i.e., in thestructure above, oligo(N) has between about 12 and about 50nucleomonomers); or between about 15 and about 35; or more particularlybetween about 20 and about 30; or even between about 21 and about 25.

In one embodiment, X is a sequence of about 4 to about 20 nucleomonomerswhich form a loop, wherein the nucleomonomers are selected from thegroup consisting of G and A.

In one embodiment, two of the Ns are unlinked, i.e., there is nophosphodiester bond between the two nucleomonomers. In one embodiment,the unlinked Ns are not in the antisense sequence.

In one embodiment, the nucleotide sequence of the loop is GAAA.

In another aspect, the invention pertains to a double-strandedoligonucleotide composition having the structure depicted in FIG. 2,where (1) N is a nucleomonomer in complementary oligonucleotide strandsof equal length where the sequence of Ns corresponds to a target genesequence; and (2) Z is a nucleomonomer in complementary oligonucleotidestrands of between about 2 and about 8 nucleomonomers in length andwhere the sequence of Z optionally corresponds to the target sequence;and (3) where M is a nucleomonomer in complementary oligonucleotidestrands of between about 2 and about 8 nucleomonomers in length andwhere the sequence of Ms optionally corresponds to the target sequence.Although the sequences of N nucleomonomers should be of the same length,the sequences of Z and M nucleomonomers may optionally be of the samelength. The invention further includes compositions such as reactionmixtures comprising such double-stranded oligonucleotides, as well asmethods for using such double-stranded oligonucleotides.

In one embodiment, Z and M are nucleomonomers selected from the groupconsisting of C and G.

In one embodiment, the sequence of Z or M is CC, GG, CG, GC, CCC, GGG,CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG,CGGG, GCCC, GGCC, or CCGG.

In another aspect, the invention pertains to a double-strandedoligonucleotide composition having the structure depicted in FIG. 3,where (1) N is a nucleomonomer in complementary oligonucleotide strandsof equal length and where the sequence of Ns corresponds to a targetgene sequence and (2) X is selected from the group consisting ofnothing; 1-20 nucleotides of 5′ overhang; 1-20 nucleotides of 3′overhang. The invention further includes compositions such as reactionmixtures comprising such double-stranded oligonucleotides, as well asmethods for using such double-stranded oligonucleotides.

In some embodiments, X is a loop structure consisting of from about 4 toabout 20 nucleomonomers, where the nucleomonomers are selected from thegroup consisting of G and A.

In the structure above, M is a nucleomonomer in complementaryoligonucleotide strands of between about 2 and about 8 nucleomonomers inlength which optionally correspond to the target sequence. In oneembodiment, M is a nucleomonomer selected from the group consisting ofcontain C and G.

In one embodiment, the sequence of M is CC, GG, CG, GC, CCC, GGG, CGG,GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG,GCCC, GGCC, or CCGG.

In another aspect, the invention pertains to a double-strandedoligonucleotide composition having the structure depicted in FIG. 4,where (1) N is a nucleomonomer in complementary oligonucleotide strandsof equal length and which correspond to a target gene sequence and (2) Yis selected from the group consisting of nothing; 1-20 nucleotides of 5′overhang; 1-20 nucleotides of 3′ overhang; a loop consisting of asequence of from about 4 to about 20 nucleomonomers, where thenucleomonomers are all either G's or A's and (3) where Z is anucleomonomer in complementary oligonucleotide strands of between about2 and about 8 nucleomonomers in length and which comprise a sequencewhich can optionally correspond to the target sequence. The inventionfurther includes compositions such as reaction mixtures comprising suchdouble-stranded oligonucleotides, as well as methods for using suchdouble-stranded oligonucleotides.

In one embodiment, Zs are nucleomonomers selected from the groupconsisting of C and G.

In one embodiment, the sequence of Z is CC, GG, CG, GC, CCC, GGG, CGG,GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG,GCCC, GGCC, or CCGG.

In another aspect, the invention pertains to a method of regulating geneexpression in a cell, comprising forming a double-strandedoligonucleotide composition as described herein and contacting a cellwith the double-stranded duplex, to thereby regulate gene expression ina cell.

In one embodiment, the invention pertains to a method of increasing thenuclease resistance of an antisense sequence, comprising forming adouble-stranded oligonucleotide composition as described herein, suchthat a double-stranded duplex is formed, wherein the nuclease resistanceof the antisense sequence is increased compared to a double-stranded,unmodified RNA molecule.

In other embodiments, the invention includes methods for introducing oneor more double-stranded nucleic acid molecule into cells (e.g.,eukaryotic cells). In particular embodiments, such methods include thosecomprising contacting cells (e.g., eukaryotic cells) with one or moredouble-stranded nucleic acid molecule. In more specific embodiments, atleast the first two (e.g., the first two, the first three, the firstfour, the first five, etc.) nucleotides at the 5′ terminus of the firststrand of the one or more double-stranded nucleic acid molecule arechemically modified at the 2′ positions and/or at least the first twonucleotides at the 5′ terminus of the second strand of the one or moredouble-stranded nucleic acid molecule are chemically modified at the 2′positions. In additional specific embodiments, the double-strandednucleic acid molecule may be between 18 and 30, between 20 and 30, orbetween 22 and 30 nucleosides in length. Further, the double-strandednucleic acid molecule may be 25 nucleosides in length. The inventionfurther includes compositions (e.g., double-stranded nucleic acidmolecule, reaction mixtures, etc.) used in these methods.

In various embodiments of the invention (e.g., those described in thepreceding paragraph), the double-stranded nucleic acid molecule maycontain an overhang (e.g., a 3′ overhang and/or a 5′ overhang) of atleast one (e.g., one, two, three, four, five, etc.) nucleoside on atleast one end (e.g., one end or both ends). Additionally, thenucleosides of the overhang(s) may be deoxy nucleosides such as deoxy-T.In specific embodiments, the overhang(s) may be or contain deoxy T-deoxyT.

Further, when nucleic acid molecules in compositions of the invention orused in methods of the invention are chemically modified at one or more2′ position, the 2′ chemical modification(s) may be a 2′-O-methylmodification, a 2′-O-propyl modification, a 2′-O-ethyl modification, a2′-fluoro modification, or a combination of such modifications.Additionally, 2′ chemical modification on such nucleic acid moleculesmay be located on ribose sugars, deoxyribose sugars, or a combination ofthese sugars.

Further, nucleic acid molecules of the invention may be combined withone or more transfection reagent to form a composition. Additionally,such compositions may be contacted with cells (e.g., eukaryotic cells).Transfection reagents used in such methods and compositions may compriseone or more cationic lipid. One example of a transfection reagent whichmay be used in the practice of the invention is LIPOFECTAMINE 2000™. Theinvention thus includes methods which employ and compositions whichcontain transfection reagents.

The invention additionally includes methods by which nucleic acidmolecules (e.g., double-stranded nucleic acid molecules) may beintroduced into eukaryotic cells by electroporation.

Further, when double-stranded nucleic acid molecules (e.g.,double-stranded RNA molecules) are used in the practice of theinvention, one strand of these double-stranded nucleic acid moleculesmay be complementary to all or part of the nucleotide sequence of an RNA(e.g., an mRNA) which is expressed in a cell (e.g., a eukaryotic cellinto which the double-stranded nucleic acid molecules are introduced).When the RNA is a mRNA, introduction of double-stranded nucleic acidmolecules in cells may inhibit expression of protein from such RNA.Thus, the invention includes methods for inhibiting gene expression.

Methods of stabilizing oligonucleotides, particularly antisenseoligonucleotides, by formation of a oligonucleotide compositionscomprising at least 3 different oligonucleotides, are disclosed inco-pending application U.S. application Ser. No. 10/357,826. Thisapplication and all of its teachings is hereby expressly incorporatedherein by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A depiction of an exemplary composition.

FIG. 2 A depiction of an exemplary composition.

FIG. 3 A depiction of an exemplary composition.

FIG. 4 A depiction of an exemplary composition.

FIG. 5 A depiction of an exemplary composition. (SEQ ID NO:1)

FIG. 6 Shows an exemplary high affinity nucleomonomer.

FIG. 7 A depiction of an exemplary composition.

FIG. 8 A depiction of an exemplary composition.

FIG. 9 A depiction of an exemplary composition. (SEQ ID NO:13)

FIG. 10 A depiction of an exemplary composition.

FIG. 11 A depiction of an exemplary composition.

FIG. 12 A depiction of an exemplary composition.

FIG. 13 A depiction of an exemplary 2 subunit morpholinooligonucleotide.

FIG. 14 shows that the length of double-stranded oligonucleotides andthe presence or absence of overhangs has no effect on function.

FIG. 14B shows the effect of structural changes on the efficacy ofsiRNAs targeting β-3-Integrin.

FIG. 15 shows that there is no correlation was observed between thelength of the double-stranded oligonucleotide and the level of PKRinduction for the given sequences.

FIG. 15B shows effect of β-3-Integrin targeted 21-mers and 27-mers onPKR expression in HMVEC Cells.

FIG. 16 shows the effect of 5′ or 3′ modification on activity ofdouble-stranded RNA duplexes.

FIG. 17 shows the effect of the size of the modifying group on activityof the double-stranded RNA duplex.

FIG. 18 shows the results of 2′-O-methyl (also referred to herein as“2′-O-Me”) modifications on the activity of double-stranded RNAduplexes.

FIG. 19 shows the inhibition of p53 by 32- and 37-mer blunt-end siRNAs.

FIG. 20 provides a schematic representation of a system for providing aproduct to a party such as a customer/purchaser.

FIG. 21 provides a schematic representation of a system for advising aparty as to the availability of a product.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention advances the prior art by providingdouble-stranded oligonucleotide compositions for use, both in vitro andin vivo, e.g., therapeutically, and by providing methods of making andusing the double-stranded antisense oligomer compositions.

Double-Stranded Oligonucleotide Compositions

Double-stranded oligonucleotides of the invention are capable ofinhibiting the synthesis of a target protein, which is encoded by atarget gene. The target gene can be endogenous or exogenous (e.g.,introduced into a cell by a virus or using recombinant DNA technology)to a cell. As used herein, the term “target gene” includespolynucleotides comprising a region that encodes a polypeptide orpolynucleotide region that regulates replication, transcription,translation, or other process important in expression of the targetprotein; or a polynucleotide comprising a region that encodes the targetpolypeptide and a region that regulates expression of the targetpolypeptide; or non-coding regions such as the 5′ or 3′ UTR or introns.Accordingly, the term “target gene” as used herein may refer to, forexample, an mRNA molecule produced by transcription from a gene ofinterest. Furthermore, the term “correspond,” as in “an oligomercorresponds to a target gene sequence,” means that the two sequences arecomplementary or homologous or bear such other biologically rationalrelationship to each other (e.g., based on the sequence ofnucleomonomers and their base-pairing properties).

The “target gene” to which an RNA molecule of the invention is directedmay be associated with a pathological condition. For example, the genemay be a pathogen-associated gene, e.g., a viral gene, atumor-associated gene, or an autoimmune disease-associated gene. Thetarget gene may also be a heterologous gene expressed in a recombinantcell or a genetically altered organism. By determining or modulating(e.g., inhibiting) the function of such a gene, valuable information andtherapeutic benefits in medicine, veterinary medicine, and biology maybe obtained.

The term “oligonucleotide” includes two or more nucleomonomerscovalently coupled to each other by linkages (e.g., phosphodiesters) orsubstitute linkages. In one embodiment, it may be desirable to use asingle-stranded nucleic acid molecule which forms a duplex structure(e.g., as described in more detail below). For example, in oneembodiment, the oligonucleotide can include a nick in either the senseof the antisense sequence.

The term “antisense” refers to a nucleotide sequence that is invertedrelative to its normal orientation for transcription and so expresses anRNA transcript that is complementary to a target gene mRNA moleculeexpressed within the host cell (e.g., it can hybridize to the targetgene mRNA molecule through Watson-Crick base pairing). An antisensestrand may be constructed in a number of different ways, provided thatit is capable of interfering with the expression of a target gene. Forexample, the antisense strand can be constructed by inverting the codingregion (or a portion thereof) of the target gene relative to its normalorientation for transcription to allow the transcription of itscomplement, (e.g., RNAs encoded by the antisense and sense gene may becomplementary). Furthermore, the antisense oligonucleotide strand neednot have the same intron or exon pattern as the target gene, andnoncoding segments of the target gene may be equally effective inachieving antisense suppression of target gene expression as codingsegments.

Accordingly, one aspect of the invention is a method of inhibiting theactivity of a target gene by introducing an RNAi, also referred to asRNA interference, agent into a cell, such that the dsRNA component ofthe RNAi agent is targeted to the gene. In one embodiment, an RNAoligonucleotide molecule may contain at least one nucleomonomer that isa modified nucleotide analogue. The nucleotide analogues may be locatedat positions where the target-specific activity, e.g., the RNAimediating activity is not substantially effected, e.g., in a region atthe 5′-end or the 3′-end of the double-stranded molecule, where theoverhangs may be stabilized by incorporating modified nucleotideanalogues.

In another aspect, double-stranded RNA molecules known in the art can beused in methods of the present invention. Double-stranded RNA moleculesknown in the art may also be modified according to the teachings hereinin conjunction with such methods, e.g., by using modifiednucleomonomers. For example, see U.S. Pat. No. 6,506,559; U.S.2002/0,173,478 A1; U.S. 2002/0,086,356 A1; Shuey, et al., “RNAi:gene-silencing in therapeutic intervention.” Drug Discov. Today 2002Oct. 15; 7(20):1040-6; Aoki, et al., “Clin. Exp. Pharmacol. Physiol.2003 January; 30(1-2):96-102; Cioca, et al., “RNA interference is afunctional pathway with therapeutic potential in human myeloid leukemiacell lines. Cancer Gene Ther. 2003 February; 10(2): 125-33.

Further examples of double-stranded RNA molecules include thosedisclosed in the following references: Kawasaki, et al., “Short hairpintype of dsRNAs that are controlled by tRNA(Val) promoter significantlyinduce RNAi-mediated gene silencing in the cytoplasm of human cells.”Nucleic Acids Res. 2003 Jan. 15; 31(2):700-7; Cottrell, et al., “Silenceof the strands: RNA interference in eukaryotic pathogens.” TrendsMicrobiol. 2003 January; 11(1):37-43; Links, “Mammalian RNAi for themasses.” Trends Genet. 2003 January; 19(1):9-12; Hamada, et al.,“Effects on RNA interference in gene expression (RNAi) in culturedmammalian cells of mismatches and the introduction of chemicalmodifications at the 3′-ends of siRNAs.” Antisense Nucleic Acid DrugDev. 2002 October; 12(5):301-9; Links, “RNAi and related mechanisms andtheir potential use for therapy.” Curr. Opin. Chem. Biol. 2002 December;6(6):829-34; Kawasaki, et al., “Short hairpin type of dsRNAs that arecontrolled by tRNA(Val) promoter significantly induce RNAi-mediated genesilencing in the cytoplasm of human cells.” Nucleic Acids Res. 2003 Jan.15; 31(2):700-7).

A nick is two non-linked nucleomonomers in an oligonucleotide. A nickcan be included at any point along the sense or antisense nucleotidesequence. In a preferred embodiment, a nick is in the sense sequence. Inanother preferred embodiment, the nick is at least about fournucleomonomers in from an end of the duplexed region of theoligonucleotide (e.g., is at least about four nucleomonomers away fromthe 5′ or 3′ end of the oligonucleotide or away from a loop structure.For example, in one embodiment, the nick is present in the middle of thesense strand of the duplex molecule (e.g., if the sense sequence of theduplex is 30 nucleomonomers in length, nucleomonomers 14 and 15 or 15and 16 are unlinked). In an embodiment, a nick may optionally be ligatedto form a circular nucleic acid molecule.

For example, in the structure shown in FIG. 5, the indicated Unucleomonomer is not bonded to the neighboring nucleomonomer, e.g., by aphosphodiester bond. The 5′ OH of the nick may optionally bephosphorylated to allow enzymatic ligation of the oligonucleotide into acircle.

As used herein, the term “nucleotide” includes any monomeric unit of DNAor RNA containing a sugar moiety (pentose), a phosphate, and anitrogenous heterocyclic base. The base is usually linked to the sugarmoiety via the glycosidic carbon (at the 1′ carbon of pentose) and thatcombination of base and sugar is called a “nucleoside.” The basecharacterizes the nucleotide with the four customary bases of DNA beingadenine (A), guanine (G), cytosine (C) and thymine (T). Inosine (I) isan example of a synthetic base that can be used to substitute for any ofthe four, naturally-occurring bases (A, C, G, or T). The four RNA basesare A, G, C, and uracil (U). Accordingly, an oligonucleotide may be anucleotide sequence comprising a linear array of nucleotides connectedby phosphodiester bonds between the 3′ and 5′ carbons of adjacentpentoses. Other modified nucleosides/nucleotides are described hereinand may also be used in the oligonucleotides of the invention.

Oligonucleotides may comprise, for example, oligonucleotides,oligonucleosides, polydeoxyribonucleotides (containing2′-deoxy-D-ribose) or modified forms thereof, e.g., DNA,polyribonucleotides (containing D-ribose or modified forms thereof),RNA, or any other type of polynucleotide which is an N-glycoside orC-glycoside of a purine or pyrimidine base, or modified purine orpyrimidine base. The term oligonucleotide includes compositions in whichadjacent nucleomonomers are linked via phosphorothioate, amide or otherlinkages (e.g., Neilsen, P. E., et al. 1991. Science. 254:1497).Generally, the term “linkage” refers to any physical connection,preferably covalent coupling, between two or more nucleic acidcomponents, e.g., catalyzed by an enzyme such as a ligase.

In addition to its art-recognized meaning (e.g., a relatively shortlength single or double-stranded sequences of deoxyribonucleotides orribonucleotides linked via phosphodiester bonds), the term“oligonucleotide” includes any structure that serves as a scaffold orsupport for the bases of the oligonucleotide, where the scaffold permitsbinding to the target nucleic acid molecule in a sequence-dependentmanner.

Oligonucleotides of the invention are isolated. The term “isolated”includes nucleic acid molecules which are synthesized (e.g., chemically,enzymatically, or recombinantly) or are naturally occurring butseparated from other nucleic acid molecules which are present in anatural source of the nucleic acid. Preferably, a naturally occurring“isolated” nucleic acid molecule is free of sequences which naturallyflank the nucleic acid molecule (i.e., sequences located at the 5′ and3′ ends of the nucleic acid molecule) in a nucleic acid molecule in anorganism from which the nucleic acid molecule is derived.

The term “nucleomonomer” includes a single base covalently linked to asecond moiety. Nucleomonomers include, for example, nucleosides andnucleotides. Nucleomonomers can be linked to form oligonucleotides thatbind to target nucleic acid sequences in a sequence specific manner.

In one embodiment, modified (non-naturally occurring) nucleomonomers canbe used in the oligonucleotides described herein. For example,nucleomonomers may be based on bases (purines, pyrimidines, andderivatives and analogs thereof) bound to substituted and unsubstitutedcycloalkyl moieties, e.g., cyclohexyl or cyclopentyl moieties, andsubstituted and unsubstituted heterocyclic moieties, e.g., 6-membermorpholino moieties or, preferably, sugar moieties.

Sugar moieties include natural, unmodified sugars, e.g., monosaccharides(such as pentoses, e.g., ribose, deoxyribose), modified sugars and sugaranalogs. Possible modifications of nucleomonomers, particularly of asugar moiety, include, for example, replacement of one or more of thehydroxyl groups with a halogen, a heteroatom, an aliphatic group, or thefunctionalization of the hydroxyl group as an ether, an amine, a thiol,or the like. One particularly useful group of modified nucleomonomersare 2′-O-methyl nucleotides, especially when the 2′-O-methyl nucleotidesare used as nucleomonomers in the ends of the oligomers. Such 2′O-methylnucleotides may be referred to as “methylated,” and the correspondingnucleotides may be made from unmethylated nucleotides followed byalkylation or directly from methylated nucleotide reagents. Modifiednucleomonomers may be used in combination with unmodifiednucleomonomers. For example, an oligonucleotide of the invention maycontain both methylated and unmethylated nucleomonomers.

Some exemplary modified nucleomonomers include sugar- orbackbone-modified ribonucleotides. Modified ribonucleotides may containa nonnaturally occurring base (instead of a naturally occurring base)such as uridines or cytidines modified at the 5-position, e.g.,5-(2-amino)propyl uridine and 5-bromo uridine; adenosines and guanosinesmodified at the 8-position, e.g., 8-bromo guanosine; deaza nucleotides,e.g., 7-deaza-adenosine; and N-alkylated nucleotides, e.g., N6-methyladenosine. Also, sugar-modified ribonucleotides may have the 2′-OH groupreplaced by a H, alxoxy (or OR), R or alkyl, halogen, SH, SR, amino(such as NH₂, NHR, NR₂,), or CN group, wherein R is lower alkyl,alkenyl, or alkynyl.

Modified ribonucleotides may also have the phosphoester group connectingto adjacent ribonucleotides replaced by a modified group, e.g., ofphosphothioate group. More generally, the various nucleotidemodifications may be combined.

In one embodiment, sense oligomers may have 2′ modifications on the ends(1 on each end, 2 on each end, 3 on each end, and 4 on each end, and soon; as well as 1 on one end, 2 on one end, 3 on one end, and 4 on oneend, and so on; and even unbalanced combinations such as 1 on one endand 2 on the other end, and so on). Likewise, the antisense strand mayhave 2′ modifications on the ends (1 on each end, 2 on each end, 3 oneach end, and 4 on each end, and so on; as well as 1 on one end, 2 onone end, 3 on one end, and 4 on one end, and so on; and even unbalancedcombinations such as 1 on one end and 2 on the other end, and so on). Inpreferred aspects, such 2′-modifications are in the sense RNA strand orthe sequences other than the antisense strand.

To further maximize endo- and exonuclease resistance, in addition to theuse of 2′ modified nucleomonomers in the ends, inter-nucleomonomerlinkages other than phosphodiesters may be used. For example, such endblocks may be used alone or in conjunction with phosphothioate linkagesbetween the 2′-O-methyl linkages. Preferred 2′-modified nucleomonomersare 2′-modified C and U bases.

Although the antisense strand may be substantially identical to at leasta portion of the target gene (or genes), at least with respect to thebase pairing properties, the sequence need not be perfectly identical tobe useful, e.g., to inhibit expression of a target gene's phenotype.Generally, higher homology can be used to compensate for the use of ashorter antisense gene. In some cases, the antisense strand generallywill be substantially identical (although in antisense orientation) tothe target gene.

One particular example of a composition of the invention has end-blockson both ends of a sense oligonucleotide and only the 3′ end of anantisense oligonucleotide. Without wishing to be bound by theory, theinventors believe that a 2′-O-modified sense strand works less well thanunmodified because it is not efficiently unwound. Accordingly, anotherembodiment of the invention includes duplexes in whichnucleomonomer-nucleomonomer mismatches are present in a sense2′-O-methyl strand (and are thought to be easier to unwind).

Accordingly, for a given first oligonucleotide strand, a number ofcomplementary second oligonucleotide strands are permitted according tothe invention. For example, in the following Tables, a targeted and anon-targeted oligonucleotide are illustrated with several possiblecomplementary oligonucleotides. The individual nucleotides may be 2′-OHRNA nucleotides (R) or the corresponding 2′-O-methyl nucleotides (M),and the oligonucleotides themselves may contain mismatched nucleotides(lower case letters).

Targeted Oligonucleotide: First Strand: CCCUUCUGUCUUGAACAUGAG (SEQ IDNO: 2) Second Strand: CTgATGTTCAAGACAGAAcGG (SEQ ID (methyl groups →)MMMMMMMMMMMMMMMMMMMMM NO: 3) CTgATGTTCAAGACAGAAcGG (SEQ IDRRRRRRRRRRRRRRRRRRRDD NO: 4) CTCAUGUUCAAGACAGAAGGG (SEQ IDRRRRRRMMMMMMMMMRRRRRR NO: 5) CTCAUGUUCAAGACAGAAGGG (SEQ IDMMMMMMRRRRRRRRRMMMMMM NO: 6) CTCAUGUUCAAGACAGAAGGG (SEQ IDRMRMRMRMRMRMRMRMRMRMR NO: 7)

Non-Targeted Oligonucleotide: First Strand: GAGTACAAGTTCTGTCTTCCC(SEQ ID NO: 8) Second Strand: GGcAAGACAGAACTTGTAgTC (SEQ ID(methyl groups →) MMMMMMMMMMMMMMMMMMMMM NO: 9) GGGAAGACAGAACTTGTACTC(SEQ ID RRRRRRMMMMMMMMMRRRRRR NO: 10) GGGAAGACAGAACTTGTACTC (SEQ IDMMMMMMRRRRRRRRRMMMMMM NO: 11) GGGAAGACAGAACTTGTACTC (SEQ IDRMRMRMRMRMRMRMRMRMRMR NO: 12)

Another example of further modifications that may be used in conjunctionwith 2′-O-methyl nucleomonomers are modification of the sugar residuesthemselves, for example alternating modified and unmodified sugars,particularly in the sense strand.

The invention further includes double stranded nucleic acid molecules(e.g., RNA molecules) which have structures defined by the followingformula:

First Strand X₁₅₋₃₀ Second Strand A₀₋₂₅X₀₋₂₅B₀₋₂₅

In the formula set out above, X, A, and B are nucleotides (e.g., A, G,C, U, etc.). Also, either of the first strand or the second strand maybe a sense strand. As a results, either of the first strand or thesecond strand may be an antisense strand. Further, X is typically anucleotide which has no modifications on the base or sugar. Further, Aand/or B are nucleotides which may independently contain one or morebase or sugar modifications. These modifications may be anymodifications known in the art or described elsewhere herein. Examplesof sugar modifications include ribose modifications at the 2′ positionsuch as 2′-O-propyl (P), 2′-O-methyl (M), 2′-O-ethyl (E), and 2′-fluoro(F). Generic examples of nucleic acid molecules of the invention includethose with the following:

    XXXXXXXXXXXXXXX XXXXX     AXXXXXXXXXXXXXX XXXXB     XXXXXXXXXXXXXXXXXXXX     AAXXXXXXXXXXXXX XXXBB     XXXXXXXXXXXXXXX XXXXX    AAAXXXXXXXXXXXX XXBBB     XXXXXXXXXXXXXXX XXXXX     AAAAXXXXXXXXXXXXBBBB     XXXXXXXXXXXXXXX XXXXX     AAAAXXXXXXXXXXX XXXBB    XXXXXXXXXXXXXXX XXXXX     AAXXXXXXXXXXXXX BBBBB     XXXXXXXXXXXXXXXXXXXX     AAAAAAAAAAAAAAA AAAAA     XXXXXXXXXXXXXXX XXXXX    AAAAAAAXXXBBBBBB BBBB

Examples of nucleic acid molecules of the invention which containspecific modifications include those with the following modifications,in which X represents an unmodified nucleotide, P represents2′-O-propyl, M represents 2′-O-methyl, E represents 2′-O-ethyl, and Frepresents 2′-fluoro:

   XXXXXXXXXXXXXXXXXXXXXXX XX    PPMMXXXXXXXXXXXXXXXXEEM MM   XXXXXXXXXXXXXXXXXXXXXXX XX    EEEEXXXXXXXXXXXXXXXXEEM MM   XXXXXXXXXXXXXXXXXXXXXXX XX    PPEEXXXXXXXXXXXXXXXXEEM MM   XXXXXXXXXXXXXXXXXXXXXXX XX    EEEEEXXXXXXXXXXXXXXXEEEE E   XXXXXXXXXXXXXXXXXXXXXXX XX    PPPPPPPXXXXXXXXXXXPPPPPPP   XXXXXXXXXXXXXXXXXXXXXXX XX    FFPPPXXXXXXXXXXXXXXXPPPFF   XXXXXXXXXXXXXXXXXXXXXXX XX    MPPPPPPPPPPPPPPPPXXXPPPPM   XXXXXXXXXXXXXXXXXXXXXXX XX    FFFFFXXXXXXXXXXXXXXXFFFFF   XXXXXXXXXXXXXXXXXXXXXXX XX    PEEPEEMPXXXXXXXXXPMEEPEEP  XXXXXXXXXXXXXXXXXXXXXXX XX    MEXXXXXXXXXXXXXXMMMMM MMMM   XXXXXXXXXXXXXXXXXXXXXXX XX    MXXXXXXXXXXXXXXXMMMMM MMMM   XXXXXXXXXXXXXXXXXXXXXXX XX    EEXXXXXXXXXXXXXXXEEEEEEE E

In some embodiments, the length of the sense strand can be 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides. Similarly, the lengthof the antisense strand can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,19, or 18 nucleotides. Further, when a double-stranded nucleic acidmolecule is formed from such sense and antisense molecules, theresulting duplex may have blunt ends or overhangs of 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides on one end orindependently on each end. Further, double stranded nucleic acidmolecules of the invention may be composed of a sense strand and anantisense strand wherein these strands are of lengths described above,and are of the same or different lengths, but share only 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 nucleotides of sequence complementarity.By way of illustration, in a situation where the sense strand is 20nucleotides in length and the antisense is 25 nucleotides in length andthe two strands share only 15 nucleotides of sequence complementarity, adouble stranded nucleic acid molecules may be formed with a 10nucleotide overhang on one end and a 5 nucleotide overhang on the otherend.

Double-stranded oligonucleotides of the invention include STEALTH™ RNAswhich may be obtained from either Sequitur Inc. (Natick, Mass.),recently acquired by Invitrogen Corporation (Carlsbad, Calif.) orInvitrogen Corporation directly. STEALTH™ RNAs are often synthesizedbased upon nucleotide sequence information provided by purchasers. Inparticular instances, purchasers may provide the nucleotide sequence ofan RNA transcript for which “knockdown” is desired and InvitrogenCorporation then selects suitable STEALTH™ RNA for the particularapplication or purchasers may provide the actual sequence of theSTEALTH™ RNAs to be used in the “knockdown” process. Typically, in thesecond instance, the nucleotide sequences provided by purchasers arebetween 20 and 30 nucleotides in length. A more detailed description ofbusiness method aspects of the invention is set out elsewhere herein.However, these business methods typically include, in part, providingSTEALTH™ RNA, as well as protocols and additional reagents and compoundsfor purchasers to use the purchased STEALTH™ RNA for knocking down geneexpression.

As a further example, the use of 2′-O-methyl RNA may be usedbeneficially in circumstances in which it is desirable to minimizecellular stress responses. RNA having 2′-O-methyl nucleomonomers may notbe recognized by cellular machinery that is thought to recognizeunmodified RNA. The use of 2′-O-methylated or partially 2′-O-methylatedRNA may avoid the interferon response to double-stranded nucleic acids,while maintaining target RNA inhibition. This RNAi (“stealth RNA”) isuseful, for example, for avoiding the interferon or other cellularstress responses, both in short RNAi (e.g., siRNA) sequences that inducethe interferon response, and in longer RNAi sequences that may inducethe interferon response.

An especially advantageous use of the present invention is in genefunction studies in which multiple RNAi sequences are used. According topresent methods known in the art, frequently there is no way ofpredicting which nucleic acid sequences might induce a stress response,including the interferon response, and in this regard the presentinvention significantly advances the state of the art. For example, ifall of the multiple sequences are partially 2′-O-methylated, the stressresponse, including interferon response, may be avoided, and thus avoidconfounding results in which some sequences affect cellular phenotypeindependent of the target gene inhibition. Other chemical modificationsin addition to 2′-O-methylation may also achieve this effect.

For example, modified sugars include D-ribose, 2′-O-alkyl (including2′-O-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino, 2′-S-alkyl,2′-halo (including 2′-fluoro), 2′-methoxyethoxy, 2′-allyloxy(—OCH₂CH═CH₂), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, andcyano and the like. In one embodiment, the sugar moiety can be a hexoseand incorporated into an oligonucleotide as described (Augustyns, K., etal., Nucl. Acids. Res. 18:4711 (1992)). Exemplary nucleomonomers can befound, e.g., in U.S. Pat. No. 5,849,902, incorporated by referenceherein.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In certain embodiments, a straight chain or branched chain alkyl has 6or fewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain,C₃-C₆ for branched chain), and more preferably 4 or fewer. Likewise,preferred cycloalkyls have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₁-C₆ includes alkyl groups containing 1 to 6 carbonatoms.

Moreover, unless otherwise specified, the term alkyl includes both“unsubstituted alkyls” and “substituted alkyls,” the latter of whichrefers to alkyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “alkylaryl” or an “arylalkyl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes the side chains of natural and unnatural amino acids. Theterm “n-alkyl” means a straight chain (i.e., unbranched) unsubstitutedalkyl group.

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond. For example, the term “alkenyl”includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl,butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.),branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups(cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. In certainembodiments, a straight chain or branched chain alkenyl group has 6 orfewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). Likewise, cycloalkenyl groups may have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₂-C₆ includes alkenyl groupscontaining 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkenyl includes both“unsubstituted alkenyls” and “substituted alkenyls,” the latter of whichrefers to alkenyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkyl groups, alkynylgroups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond. For example, the term “alkynyl”includes straight-chain alkynyl groups (e.g., ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.),branched-chain alkynyl groups, and cycloalkyl or cycloalkenylsubstituted alkynyl groups. In certain embodiments, a straight chain orbranched chain alkynyl group has 6 or fewer carbon atoms in its backbone(e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). The termC₂-C₆ includes alkynyl groups containing 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkynyl includes both“unsubstituted alkynyls” and “substituted alkynyls,” the latter of whichrefers to alkynyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkyl groups, alkynylgroups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withindependently selected groups such as alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.Examples of halogen substituted alkoxy groups include, but are notlimited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻(with an appropriate counterion).

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The term “substituted” includes independently selected substituentswhich can be placed on the moiety and which allow the molecule toperform its intended function. Examples of substituents include alkyl,alkenyl, alkynyl, aryl, (CR′R″)₀₋₃NR′R″, (CR′R″)₀₋₃CN, NO₂, halogen,(CR′R″)₀₋₃C(halogen)₃, (CR′R″)₀₋₃CH(halogen)₂, (CR′R″)₀₋₃CH₂(halogen),(CR′R″)₀₋₃CONR′R″, (CR′R″)₀₋₃S(O)₁₋₂NR′R″, (CR′R″)₀₋₃CHO,(CR′R″)₀₋₃O(CR′R″)₀₋₃H, (CR′R″)₀₋₃S(O)₀₋₂R′, (CR′R″)₀₋₃O(CR′R″)₀₋₃H,(CR′R″)₀₋₃COR′, (CR′R″)₀₋₃CO₂R′, or (CR′R″)₀₋₃OR′ groups; wherein eachR′ and R″ are each independently hydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl,C₂-C₅ alkynyl, or aryl group, or R′ and R″ taken together are abenzylidene group or a —(CH₂)₂O(CH₂)₂— group.

The term “amine” or “amino” includes compounds or moieties in which anitrogen atom is covalently bonded to at least one carbon or heteroatom.The term “alkyl amino” includes groups and compounds wherein thenitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl,” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “base” includes the known purine and pyrimidine heterocyclicbases, deazapurines, and analogs (including heterocyclic substitutedanalogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g., 1-alkyl-,1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomersthereof. Examples of purines include adenine, guanine, inosine,diaminopurine, and xanthine and analogs (e.g., 8-oxo-N⁶-methyladenine or7-diazaxanthine) and derivatives thereof. Pyrimidines include, forexample, thymine, uracil, and cytosine, and their analogs (e.g.,5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil,5-(1-propynyl)cytosine and 4,4-ethanocytosine). Other examples ofsuitable bases include non-purinyl and non-pyrimidinyl bases such as2-aminopyridine and triazines.

In a preferred embodiment, the nucleomonomers of an oligonucleotide ofthe invention are RNA nucleotides. In another preferred embodiment, thenucleomonomers of an oligonucleotide of the invention are modified RNAnucleotides. Thus, the oligunucleotides contain modified RNAnucleotides.

The term “nucleoside” includes bases which are covalently attached to asugar moiety, preferably ribose or deoxyribose. Examples of preferrednucleosides include ribonucleosides and deoxyribonucleosides.Nucleosides also include bases linked to amino acids or amino acidanalogs which may comprise free carboxyl groups, free amino groups, orprotecting groups. Suitable protecting groups are well known in the art(see P. G. M. Wuts and T. W. Greene, “Protective Groups in OrganicSynthesis”, 2^(nd) Ed., Wiley-Interscience, New York, 1999).

The term “nucleotide” includes nucleosides which further comprise aphosphate group or a phosphate analog.

As used herein, the term “linkage” includes a naturally occurring,unmodified phosphodiester moiety (—O—(PO₂ ⁻)—O—) that covalently couplesadjacent nucleomonomers. As used herein, the term “substitute linkage”includes any analog or derivative of the native phosphodiester groupthat covalently couples adjacent nucleomonomers. Substitute linkagesinclude phosphodiester analogs, e.g., phosphorothioate,phosphorodithioate, and P-ethyoxyphosphodiester, P-ethoxyphosphodiester,P-alkyloxyphosphotriester, methylphosphonate, and nonphosphoruscontaining linkages, e.g., acetals and amides. Such substitute linkagesare known in the art (e.g., Bjergarde et al. 1991. Nucleic Acids Res.19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47).

In certain embodiments, oligonucleotides of the invention comprise 3′and 5′ termini (except for circular oligonucleotides). In oneembodiment, the 3′ and 5′ termini of an oligonucleotide can besubstantially protected from nucleases e.g., by modifying the 3′ or 5′linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For example,oligonucleotides can be made resistant by the inclusion of a “blockinggroup.” The term “blocking group” as used herein refers to substituents(e.g., other than OH groups) that can be attached to oligonucleotides ornucleomonomers, either as protecting groups or coupling groups forsynthesis (e.g., FITC, propyl (CH₂—CH₂—CH₃), glycol(—O—CH2-CH2-O-)phosphate (PO₃ ²), hydrogen phosphonate, orphosphoramidite). “Blocking groups” also include “end blocking groups”or “exonuclease blocking groups” which protect the 5′ and 3′ termini ofthe oligonucleotide, including modified nucleotides and non-nucleotideexonuclease resistant structures.

Exemplary end-blocking groups include cap structures (e.g., a7-methylguanosine cap), inverted nucleomonomers, e.g., with 3′-3′ or5′-5′ end inversions (see, e.g., Ortiagao et al. 1992. Antisense Res.Dev. 2:129), methylphosphonate, phosphoramidite, non-nucleotide groups(e.g., non-nucleotide linkers, amino linkers, conjugates) and the like.The 3′ terminal nucleomonomer can comprise a modified sugar moiety. The3′ terminal nucleomonomer comprises a 3′-O that can optionally besubstituted by a blocking group that prevents 3′-exonuclease degradationof the oligonucleotide. For example, the 3′-hydroxyl can be esterifiedto a nucleotide through a 3′→3′ internucleotide linkage. For example,the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, andpreferably, ethoxy. Optionally, the 3′→3′ linked nucleotide at the 3′terminus can be linked by a substitute linkage. To reduce nucleasedegradation, the 5′ most 3′→5′ linkage can be a modified linkage, e.g.,a phosphorothioate or a P-alkyloxyphosphotriester linkage. Preferably,the two 5′ most 3′→5′ linkages are modified linkages. Optionally, the 5′terminal hydroxy moiety can be esterified with a phosphorus containingmoiety, e.g., phosphate, phosphorothioate, or P-ethoxyphosphate.

In one embodiment, the sense strand of an oligonucleotide comprises a 5′group that allows for RNAi activity but which renders the sense strandinactive in terms of gene targeting. Preferably, such a 5′ modifyinggroup is a phosphate group or a group larger than a phosphate group.Oligonucleotides of this type often exhibit increased specificity for atarget gene in a cell that corresponds to the nucleotide sequence of theantisense strand. This is because the sense strand in such anoligonucleotide is often rendered incapable of mediating cleavage of anynucleotide sequence it might bind to non-specifically and thus will notinactivate any other genes in the cell. Thus, observed decrease in theexpression of a gene within a cell transfected with such anoligonucleotide will often be attributed to the direct or indirecteffect of the anti-sense strand. The term “specificity for a targetgene,” as used herein means the extent to which an effect of anoligonucleotide on a cell can be attributed directly or indirectly tothe inhibition of expression of a target gene by an antisense nucleotidesequence present in said oligonucleotide.

Thus, according to another embodiment, the invention provides a methodof increasing the specificity of an oligonucleotide for a target gene ina cell, wherein said oligonucleotide comprises a sense strand and anantisense strand, wherein both the sense strand and the antisense strandare capable of binding to corresponding nucleotide sequences if presentin said cell, said method comprising the step of modifying the 5′terminal hydroxy moiety of said sense strand with a phosphate group or agroup larger than a phosphate group prior to contacting saidoligonucleotide with said cell so as to render said sense strandincapable of mediating cleavage of any nucleotide sequence it might bindto non-specifically and thus will not inactivate any other genes in thecell.

The invention also provides an improvement in a method of regulating theexpression of a target gene in a cell, comprising contacting a cell withan oligonucleotide comprising a sense strand and an antisense strand,wherein both the sense strand and the antisense strand are capable ofbinding to corresponding nucleotide sequences if present in said cell,said improvement comprising the step of modifying the 5′ terminalhydroxy moiety of said sense strand with a phosphate group or a grouplarger than a phosphate group prior to contacting said oligonucleotidewith said cell so as to render said sense strand incapable of binding tocorresponding nucleotide sequences if present in said cell.

In another embodiment, the antisense strand of an oligonucleotidecomprises a 5′ phosphate group or a group larger than a phosphate group.Oligonucleotides in accordance with this aspect of the invention whichcomprise such a modification of the antisense strand typically cannotinactivate a target gene that corresponds to the nucleotide sequence ofsaid antisense strand. However, such modified oligonucleotides areextremely useful as controls for non-specific effects caused by acorresponding oligonucleotide that lacks such a 5′ modification on theantisense strand. Non-specific effects include all effects on the cellby an oligonucleotide except those caused directly or indirectly by theinactivation of a target gene by a corresponding nucleotide sequencepresent in the antisense strand of said oligonucleotide.

Thus, according to a related embodiment, the invention provides a methodof determining the non-specific effects of a test oligonucleotidetransfected into a population of cells, wherein said testoligonucleotide comprises a sense strand, an antisense strand and atleast one modified oligonucleotide, said method comprising the steps of:a) providing an oligonucleotide having the same nucleotide sequence andmodifications as said test oligonucleotide and additionally comprising a5′ modification on the antisense strand, wherein said 5′ modification isa phosphate group or a group larger than a phosphate group; b)transfecting said population of cells with the oligonucleotide providedin step a); and c) determining the effect of the oligonucleotideprovided in step a) on said population of cells.

In particular embodiments, the invention comprises methods for measuringuptake of nucleic acid molecules (e.g., double-stranded nucleic acidmolecules) by cells (e.g., eukaryotic cells). In particular embodiments,such methods comprise: (a) contacting a cell or population of cells(e.g., eukaryotic cells) with a detectably labeled nucleic acid moleculeor a population of detectably labeled nucleic acid molecules (e.g., amixture of detectably labeled nucleic acid molecules such asdouble-stranded nucleic acid molecules which differ in nucleotidesequence) under conditions which allow for some or all of the nucleicacid molecules to enter then cell or cells; (b) quantifying the amountof detectably labeled which has entered either (i) the cell or (ii) someor all of the cells of the population, for example by exposing the cellsto one or more wavelengths of light which excite one or more label(e.g., one or more fluorescent label); and (c) measuring one or moresignal (e.g., one or more fluorescent or other signal) generated fromthe label(s) in the cells. In more specific embodiments, thedouble-stranded nucleic acid molecule may contain one or more bound(e.g., covalently bound) label (e.g., one or more fluorescent label). Inadditional specific embodiments, the double-stranded nucleic acidmolecule may be between 18 and 30, between 20 and 30, or between 22 and30 nucleosides in length. Further, the double-stranded nucleic acidmolecule may be 25 nucleosides in length.

The detectable label employed may be any suitable label known in the artand include radiolabels, chemiluminescent, and fluorescent labels. Inmany instances, the label will be of a type which does not substantiallyalter the uptake of the nucleic acid molecules to which they areattached by cells.

In particular embodiments, one or more label may be located on one orboth 3′ ends and/or on one or both 5′ ends of the double-strandednucleic acid molecules.

Additionally, the signal(s) generated by the label(s) may be measured byany number of ways, including visually (e.g., by microscopy) orfluorescent activated cell sorting (FACS). In any event, measurement ofthe signal(s) generated label(s) may be used to determine (a) the numberor percentage of cells which have taken up the label(s), (b) the amountof one or more label taken up by individual cells or groups of cells, or(c) both (a) and (b).

Labels used in methods and compositions of the invention varyconsiderably but will often be labels which are readily detectable.Examples of such labels include the fluorescent labels FITC and6-carboxyfluorescein.

Methods of the invention further include those where cells are contactedwith one or more labels for a particular period of time (e.g., one hour,two hours, three hours, four hours, five hours, six hours, seven hours,eight hours, nine hours, ten hours, from about one hour to about tenhours, from about three hours to about eight hours, from about fourhours to about seven hours, etc.) and then the cells are examined forthe presence of the label.

The invention additionally includes methods for determining the ratio ofviable to non-viable cells (e.g., eukaryotic cells) in populations ofcells. In particular aspects, such methods include those which comprise(a) contacting cells of a population with a double-stranded nucleic acidmolecule, (b) contacting the cells of the population of (a) with a dyewhich preferentially stains non-viable cells, and (c) comparing thenumber of stained cells to the number of unstained cells to arrive atthe ratio of viable to non-viable cells in the population. In specificembodiments, step (b) above may performed, ten hours, twelve hours,twenty hours, twenty-four hours, thirty hours, forty hours, forty-eighthours, from about ten hours to about forty-eight hours, from abouttwelve hours to about twenty-four hours, or from about sixteen hours toabout thirty hours after step (a).

According to another related embodiment, the invention provides a kitfor determining the non-specific effect on a cell of a testoligonucleotide transfected into said cell, wherein said testoligonucleotide comprises a sense strand, an antisense strand and atleast one modified oligonucleotide, said kit comprising a first vesselcontaining an oligonucleotide having the same nucleotide sequence andmodifications as said test oligonucleotide and additionally comprising a5′ modification on said antisense strand, wherein said 5′ modificationis a phosphate group or a group larger than a phosphate group; andinstructions for using said 5′-modified oligonucleotide to determine thenon-specific effect on said cell of said test oligonucleotidetransfected into said cell. In a preferred embodiment, the kitadditionally comprises one or more of the following: an independentvessel containing a dye which distinguishes live cells from dead cellsin said cell population; an independent vessel comprising anoligonucleotide known to inhibit a gene expressed in said cellpopulation; an independent vessel containing said test oligonucleotide;and an independent vessel comprising an oligonucleotide having the samenucleotide sequence and modifications as said test oligonucleotide andadditionally comprising a 5′ detectable end blocking group on the sensestrand.

In yet another embodiment, the invention provides an oligonucleotide ofthe invention that comprises at least one modified RNA nucleotide and adetectable moiety on one or both of the sense strand and the antisensestrand. The term “detectable moiety”, as used herein, refers to achemical moiety that renders the oligonucleotide detectable (e.g.,visibly detectable) within a cell. In many instances, the detectablemoiety is a fluorescent molecule. In some instances, the detectablemoiety is a fluorescent or chemiluminescent fluorophore. In particularinstances, the detectable moiety is FITC. In another instance, thedetectable moiety is a 5′- or 3′-end blocking group located on one orboth of the sense and the antisense strand. In one instance, at leastone RNA nucleotide in this oligonucleotide contains at least onechemical modification. These modified RNA nucleotides may contain one ormore 2′-fluoro, 2′-O-methyl, 2′-O-ethyl, and/or 2′-O-propyl groups. Inparticular instances, (1) all of the nucleotides of the antisense strandand/or the sense strand contain 2′-fluoro, 2′-O-methyl, 2′-O-ethyl,and/or 2′-O-propyl groups and (2) the antisense and/or the sense strandcontains FITC as a 3′- and/or 5′-end blocking group. In particularembodiments, the entire sense strand is 2′-fluoroated, 2′-O-methylated,2′-O-ethylated, or 2′-O-propylated and both the sense and antisensestrands contain FITC as a 3′- or 5′-end blocking group.

Oligonucleotides of the invention that comprise a detectable moiety canserve as a control for the uptake of corresponding oligonucleotideshaving the same nucleotide sequence (with or without some or all of themodifications on the nucleotides present in the blockedoligonucleotide), but lacking the detectable moiety. Oligonucleotidescomprising a detectable moiety are particularly useful in establishingoptimal transfection conditions for cells that will be treated with thecorresponding unblocked or undetectable oligonucleotide. When adetectable moiety present on the antisense strand which corresponds to atarget gene is a 5′-end blocking group, that antisense strand isincapable of inhibiting the expression of that target gene. If thedetectable moiety is present elsewhere on the antisense strand and/oranywhere on the sense strand, the oligonucleotide may still be capableof inhibiting expression of the target gene, as well as being detected.An oligonucleotide capable of inhibiting expression of a target gene isreferred to as an “active oligonucleotide.” The invention includesactive oligonucleotides and compositions comprising such activeoligonucleotides.

Some detectable moiety-containing oligonucleotides of the invention canbe detected in a cell nucleus for at least 72 hours followingtransfection. Moreover, oligonucleotides comprising a detectable moiety,when used in parallel with a corresponding active oligonucleotide (or,alternatively, if the oligonucleotide comprising a detectable moiety isitself active), provide the best control for any variation intransfection conditions and reagents on the day that transfection isperformed. Also, the persistence of these detectable oligonucleotides inthe cell is typically highly correlated with siRNA activity of acorresponding active oligonucleotide.

Thus, according to a related embodiment, the invention provides a methodof determining the uptake of a test oligonucleotide by a population ofcells using a transfection protocol, wherein the test oligonucleotidecomprises a sense strand, an antisense strand and at least one modifiedoligonucleotide, the method comprising the steps of: a) providing anoligonucleotide having (i) the same nucleotide sequence as the testoligonucleotide, (ii) the same number of and/or type of modifications, adifferent number of and/or type of modifications, or no modifications,and (iii) a detectable moiety on one or both of the sense and antisensestrands; b) using the transfection protocol to transfect said populationof cells with the oligonucleotide provided in step a); and c) usingdetecting means to determine the number of cells in said populationtransfected with oligonucleotide provided in step a). In one embodiment,the test oligonucleotide additionally comprises a detectable moiety, isan active oligonucleotide and is the oligonucleotide provided in stepa).

The term “detecting means,” as used herein, encompasses any method ofdetection that would allow one to quantitatively or qualitatively thepresence of the oligonucleotide comprising a detectable moiety within acell. For example, if the detectable moiety is a fluorescent label, thendetecting means would comprise the use of light source having awavelength to cause excitation of said fluorescent label and either afluorescent microscope fitted with a filter appropriate to observe theemission from said excited fluorescent label or a fluorescence activatedcell sorter.

According to another related embodiment, the invention provides a kitfor optimizing the uptake of a test oligonucleotide by a population ofcells, wherein said test oligonucleotide comprises a sense strand, anantisense strand and at least one modified oligonucleotide, said kitcomprising a first vessel containing an oligonucleotide having the samenucleotide sequence and modifications as said test oligonucleotide andadditionally comprising a detectable moiety on one or both of said senseand antisense strands and instructions for using said detectablemoiety-containing oligonucleotide to determine uptake of said testoligonucleotide by said population of cells. In a preferred embodiment,the kit additionally comprises one or more of the following: anindependent vessel containing a dye which distinguishes live cells fromdead cells in said cell population; an independent vessel comprising anoligonucleotide known to inhibit a gene expressed in said cellpopulation; an independent vessel containing said test oligonucleotide;and an independent vessel containing an oligonucleotide having the samenucleotide sequence and modifications as said test oligonucleotide andadditionally comprising a 5′ modification on the antisense strand,wherein said 5′ modification is a phosphate group or a group larger thana phosphate group, and said 5′ modification inactivates saidoligonucleotide.

In each of the kit embodiments, the detectable moiety is preferablyFITC. In kit embodiments that include an independent vessel containing adye that distinguishes live cells from dead cells in said cellpopulation, the dye is preferably dead red stain (Molecular Probes,Eugene Oreg., ethidium homodimer cat. no. E-1169).

In one embodiment, the oligonucleotides included in the composition arehigh affinity oligonucleotides. The term “high affinity” as used hereinincludes oligonucleotides that have a Tm (melting temperature) of orgreater than about 60° C., greater than about 65° C., greater than about70° C., greater than about 75° C., greater than about 80° C. or greaterthan about 85° C. The Tm is the midpoint of the temperature range overwhich the oligonucleotide separates from the target nucleotide sequence.At this temperature, 50% helical (hybridized) versus coil (unhybridized)forms are present. Tm is measured by using the UV spectrum to determinethe formation and breakdown (melting) of hybridization. Base stackingoccurs during hybridization, which leads to a reduction in UVabsorption. Tm depends both on GC content of the two nucleic acidmolecules and on the degree of sequence complementarity. Tm can bedetermined using techniques that are known in the art (see for example,Monia et al. 1993. J. Biol. Chem. 268:145; Chiang et al. 1991. J. Biol.Chem. 266:18162; Gagnor et al. 1987. Nucleic Acids Res. 15:10419; Moniaet al. 1996. Proc. Natl. Acad. Sci. 93:15481; Publisis and Tinoco. 1989.Methods in Enzymology 180:304; Thuong et al. 1987. Proc. Natl. Acad.Sci. USA 84:5129).

In one embodiment, an oligonucleotide can include an agent whichincreases the affinity of the oligonucleotide for its target sequence.The term “affinity enhancing agent” includes agents that increase theaffinity of an oligonucleotide for its target. Such agents include,e.g., intercalating agents and high affinity nucleomonomers.Intercalating agents interact strongly and nonspecifically with nucleicacids. Intercalating agents serve to stabilize RNA-DNA duplexes and thusincrease the affinity of the oligonucleotides for their targets.Intercalating agents are most commonly linked to the 3′ or 5′ end ofoligonucleotides. Examples of intercalating agents include acridine,chlorambucil, benzopyridoquinoxaline, benzopyridoindole,benzophenanthridine, and phenazinium. The agents may also impart othercharacteristics to the oligonucleotide, for example, increasingresistance to endonucleases and exonucleases.

In one embodiment, a high affinity nucleomonomer is incorporated into anoligonucleotide. The language “high affinity nucleomonomer” as usedherein includes modified bases or base analogs that bind to acomplementary base in a target nucleic acid molecule with higheraffinity than an unmodified base, for example, by having moreenergetically favorable interactions with the complementary base, e.g.,by forming more hydrogen bonds with the complementary base. For example,high affinity nucleomonomer analogs such as aminoethyoxy phenoxazine(also referred to as a G clamp), which forms four hydrogen bonds withguanine are included in the term “high affinity nucleomonomer.” A highaffinity nucleomonomer is illustrated in FIG. 6 (see, e.g., Flanagan, etal., 1999. Proc. Natl. Acad. Sci. 96:3513).

Other exemplary high affinity nucleomonomers are known in the art andinclude 7-alkenyl, 7-alkynyl, 7-heteroaromatic-, or7-alkynyl-heteroaromatic-substituted bases or the like which can besubstituted for adenosine or guanosine in oligonucleotides (see, e.g.,U.S. Pat. No. 5,594,121). Also, 7-substituted deazapurines have beenfound to impart enhanced binding properties to oligonucleotides, i.e.,by allowing them to bind with higher affinity to complementary targetnucleic acid molecules as compared to unmodified oligonucleotides. Highaffinity nucleomonomers can be incorporated into the oligonucleotides ofthe instant invention using standard techniques.

In another embodiment, an agent that increases the affinity of anoligonucleotide for its target comprises an intercalating agent. As usedherein, the language “intercalating agent” includes agents which canbind to a DNA double helix. When covalently attached to anoligonucleotide of the invention, an intercalating agent enhances thebinding of the oligonucleotide to its complementary genomic DNA targetsequence. The intercalating agent may also increase resistance toendonucleases and exonucleases.

Exemplary intercalating agents are taught by Helene and Thuong (1989.Genome 31:413), and include e.g., acridine derivatives (Lacoste et al.1997. Nucleic Acids Research. 25:1991; Kukreti et al. 1997. NucleicAcids Research. 25:4264); quinoline derivatives (Wilson et al. 1993.Biochemistry 32:10614); and benzo[f]quino[3,4-b]quioxaline derivatives(Marchand et al. 1996. Biochemistry. 35:5022; Escude et al. 1998. Proc.Natl. Acad. Sci. 95:3591).

Intercalating agents can be incorporated into an oligonucleotide usingany convenient linkage. For example, acridine or psoralen can be linkedto the oligonucleotide through any available —OH or —SH group, e.g., atthe terminal 5′ position of the oligonucleotide, the 2′ positions ofsugar moieties, or an OH, NH₂, COOH, or SH incorporated into the5-position of pyrimidines using standard methods.

In one embodiment, when included in an RNase H activating antisensenucleotide sequence, an agent that increases the affinity of anoligonucleotide for its target is not positioned adjacent to an RNaseactivating region of the oligonucleotide, e.g., is positioned adjacentto a non-RNase activating region. Preferably, the agent that increasesthe affinity of an oligonucleotide for its target is placed at adistance as far as possible from the RNase activating domain of thechimeric antisense sequence such that the specificity of the chimericantisense sequence is not altered when compared with the specificity ofa chimeric antisense sequence which lacks the intercalating compound. Inone embodiment, this can be accomplished by positioning the agentadjacent to a non-RNase activating region. The specificity of theoligonucleotide can be tested by demonstrating that transcription of anon-target sequence, preferably a non-target sequence which isstructurally similar to the target (e.g., has some sequence homology oridentity with the target sequence but which is not identical in sequenceto the target), is not inhibited to a greater degree by anoligonucleotide comprising an affinity enhancing agent than by anoligonucleotide directed against the same target that does not comprisean affinity enhancing agent.

Double-stranded oligonucleotides of the invention may be formed by asingle, self-complementary nucleic acid strand or two separatecomplementary nucleic acid strands. Duplex formation can occur eitherinside or outside the cell containing the target gene.

As used herein, the term “double-stranded” includes one or more nucleicacid molecules comprising a region of the molecule in which at least aportion of the nucleomonomers are complementary and hydrogen bond toform a duplex.

As used herein, the term “duplex” includes the region of thedouble-stranded nucleic acid molecule(s) that is (are) hydrogen bondedto a complementary sequence.

Double-stranded oligonucleotides of the invention may comprise anucleotide sequence that is sense to a target gene and a complementarysequence that is antisense to the target gene. The sense and antisensenucleotide sequences correspond to the target gene sequence, e.g., areidentical or are sufficiently identical to effect target gene inhibition(e.g., are about at least about 98% identical, 96% identical, 94%, 90%identical, 85% identical, or 80% identical) to the target gene sequence.

When comprised of two separate complementary nucleic acid molecules, theindividual nucleic acid molecules can be of different lengths.

In one embodiment, a double-stranded oligonucleotide of the invention isdouble-stranded over its entire length, i.e., with no overhangingsingle-stranded sequence at either end of the molecule, i.e., isblunt-ended. In another embodiment, a double-stranded oligonucleotide ofthe invention is not double-stranded over its entire length. Forinstance, when two separate nucleic acid molecules are used, one of themolecules, e.g., the first molecule comprising an antisense sequence,can be longer than the second molecule hybridizing thereto (leaving aportion of the molecule single-stranded). Likewise, when a singlenucleic acid molecule is used a portion of the molecule at either endcan remain single-stranded.

In one embodiment, a double-stranded oligonucleotide of the invention isdouble-stranded over at least about 70% of the length of theoligonucleotide. In another embodiment, a double-strandedoligonucleotide of the invention is double-stranded over at least about80% of the length of the oligonucleotide. In another embodiment, adouble-stranded oligonucleotide of the invention is double-stranded overat least about 90%-95% of the length of the oligonucleotide. In anotherembodiment, a double-stranded oligonucleotide of the invention isdouble-stranded over at least about 96%-98% of the length of theoligonucleotide.

In one embodiment, the double-stranded duplex constructs of theinvention can be further stabilized against nucleases by forming loopstructures at the 5′ or 3′ end of the sense or antisense strand of theconstruct. For example, the construct can take the form shown in FIG. 1,where the Ns are nucleomonomers in complementary oligonucleotide strands(i.e., the top N strand is complementary to the bottom N strand) ofequal length (e.g., between about 12 and about 40 nucleotides in length)and X and Y are each independently selected from a group consisting ofnothing (i.e., the construct is a blunt ended construct with no loopsand no overhang); from about 1 to about 20 nucleotides of 5′ overhang;from about 1 to about 20 nucleotides of 3′ overhang; a GAAA loop(tetra-loop); and a loop consisting from about 4 to about 20nucleomonomers (where the nucleomonomers are all either G's or A's).

The sequence of Ns corresponds to the target gene sequence (e.g., ishomologous or identical to a nucleotide sequence that is sense orantisense to the target gene sequence), while the nucleotide sequence ofthe loop structure does not correspond to the target gene sequence.

For example, such loops can comprise all G's and A's and be from about 4to about 20 nucleotides in length. In one embodiment, such a loop can bea tetra-loop having a sequence GAAA as depicted in FIG. 7.

In one embodiment, the number of Ns is about 27.

In embodiments in which loops are at one or both ends of the construct,the oligonucleotide can be divided by having a “nick” which is twonon-linked nucleomonomers at any point along the sense or antisensestrand, but preferably along the sense strand. Preferably, the nick isat least four bases from the nearest end of the duplexed region (toprovide enough thermodynamic stability).

In another embodiment, a construct of the invention can take the formdepicted in FIG. 8, where the Ns are complementary nucleomonomers inoligonucleotide strands of equal length (e.g., between 12-40nucleomonomers in length); Zs are nucleomonomers in complementaryoligonucleotide strands of between about 2 and about 8 nucleomonomers inlength and which comprise a sequence which can optionally correspond tothe target sequence; and where Ms are nucleomonomers in complementaryoligonucleotide strands of between about 2 and about 8 nucleomonomers inlength and which can optionally correspond to the target sequence.

Preferably, the Zs and Ms are nucleomonomers selected from the groupconsisting of C's and G's to make the end of the duplex morethermodynamically stable. Ends of duplexes can become single strandedtransiently, and since duplex RNA is more stable than single-strandedRNA, the enhanced stability of the duplex on the ends will result inhigher nuclease stability.

A preferred sequence for Z or M in the antisense strand is from 2-8nucleomonomers in length or preferably from 3-4 nucleomonomers inlength, e.g., (from 5′ to 3′) CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG,CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC,or CCGG. The complementary strand would have the correspondingcomplementary sequence.

In still another embodiment, a construct of the invention has the formdepicted in FIG. 3, where Ns are nucleomonomers in complementaryoligonucleotide strands (i.e., the top N strand is complementary to thebottom N strand) of equal length (e.g., from between about 12 to about40 nucleomonomers in length) and X is selected from the group consistingof nothing (i.e., leaving blunt ends with no loop or overhang); 1-20nucleotides of 5′ overhang; 1-20 nucleotides of 3′ overhang; a GAAA loop(tetra-loop); and a loop consisting of from about 4 to about 20nucleomonomers (where the nucleomonomers are all either G's or A's) andwhere Ms are nucleomonomers in complementary oligonucleotide strands ofbetween about 2 and about 8 nucleomonomers in length (which canoptionally correspond to the target sequence). Preferably, Ms arenucleomonomers selected from the group consisting of contain C's andG's.

A preferred sequence for M in the antisense strand is from 2-8nucleomonomers in length or preferably from 3-4 nucleomonomers inlength, e.g., (from 5′ to 3′) CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG,CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC,or CCGG and the corresponding complement on the opposite strand.

In another embodiment, the construct can take the form depicted in FIG.4, where Ns are nucleomonomers in complementary oligonucleotide strandsof equal length (e.g., from between about 12 to about 40 nucleomonomersin length) and Y is selected from the group consisting of nothing (i.e.,leaving blunt ends with no loop or overhang; 1-20 nucleotides of 5′overhang; 1-20 nucleotides of 3′ overhang; a GAAA loop (tetra-loop); anda loop consisting of a sequence of from about 4 to about 20nucleomonomers (where the nucleomonomers are all either Gs or A's) andwhere Zs are nucleomonomers in complementary oligonucleotide strands ofbetween about 2 and about 8 nucleomonomers in length and which comprisea sequence which can optionally correspond to the target sequence.Preferably, the Zs are nucleomonomers selected from the group consistingof Cs and Gs to make the end of the duplex more stable.

A preferred sequence for Z in the antisense strand is from 2-8nucleomonomers in length or preferably from 3-4 nucleomonomers inlength, e.g., (from 5′ to 3′) CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG,CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC orCCGG (and the corresponding complement on the opposite strand). Forexample, in the structure shown in FIG. 9, GGCC on the end (and itscomplement) confers additional stability.

The invention also relates to a double-stranded oligonucleotidecomposition having the following structure depicted in FIG. 10, wherein(1) oligoA is an oligonucleotide of a number of nucleomonomers; (2)oligoB is an oligonucleotide that has the same number of nucleomonomersas oligoA and that is complementary to oligoA; (3) either oligoA oroligoB corresponds to a target gene sequence.

In this structure, X may be selected from (a) nothing; (b) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 5′ end of oligoA and constituting a 5′ overhang; (c) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 3′ end of oligoB and constituting a 3′ overhang; (d) and anoligonucleotide of about 4 to about 20 nucleomonomers covalently bondedto the 3′ end of oligoB and the 5′ end of oligoA and constituting a loopstructure, where the nucleomonomers are selected from the groupconsisting of G and A.

Similarly, Y may be selected from (a) nothing; (b) an oligonucleotide ofabout 1 to about 20 nucleotides covalently bonded to the 5′ end ofoligoB and constituting a 5′ overhang; (c) an oligonucleotide of about 1to about 20 nucleotides covalently bonded to the 3′ end of oligoA andconstituting a 3′ overhang; (d) and an oligonucleotide of about 4 toabout 20 nucleomonomers covalently bonded to the 3′ end of oligoA andthe 5′ end of oligoB and constituting a loop structure, where thenucleomonomers are selected from the group consisting of G and A.

Similarly, the invention includes a double-stranded oligonucleotidecomposition having the structure depicted in FIG. 11, wherein (1) oligoAis 5′-(N)₁₅₋40-(M)₂₋₈-3′ and oligoB is 5′-(N)₁₅₋40-(M)₂₋₈-3′, whereineach of N and M is independently a nucleomonomer; (2) both of thesequences of Ns are complementary oligonucleotide strands of equallength having between about 15 and 40 nucleomonomers; (3) at least oneof the sequences of Ns, optionally with some or all of the flanking Ms,corresponds to a target gene sequence. Both of the sequences of Ms arecomplementary oligonucleotide strands of between about 2 and about 8nucleomonomers in length. The two M strands are optionally of the samelength.

The group X indicated by the curved line is selected from (a) nothing;(b) an oligonucleotide of about 1 to about 20 nucleotides covalentlybonded to the 5′ end of oligoA and constituting a 5′ overhang; (c) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 3′ end of oligoB and constituting a 3′ overhang; (d) and anoligonucleotide of about 4 to about 20 nucleomonomers covalently bondedto the 3′ end of oligoB and the 5′ end of oligoA and constituting a loopstructure, where the nucleomonomers are selected from the groupconsisting of G and A.

Likewise, the invention pertains to a double-stranded oligonucleotidecomposition having the structure depicted in FIG. 12, wherein (1) oligoAis 5′-(Z)₂₋₈-(N)₁₂₋₄₀-3′ and oligoB is 5′-(Z)₂₋₈-(N)₁₂₋₄₀-3′, whereineach of N and Z is independently a nucleomonomer; (2) both of thesequences of Ns are complementary oligonucleotide strands of equallength having between about 12 and 40 nucleomonomers; (3) at least oneof the sequences of Ns, optionally with some or all of the flanking Zs,corresponds to a target gene sequence. Both of the sequences of Zs arecomplementary oligonucleotide strands of between about 2 and about 8nucleomonomers in length. The two Z strands are optionally of the samelength.

Here, Y is selected from (a) nothing; (b) an oligonucleotide of about 1to about 20 nucleotides covalently bonded to the 5′ end of oligoB andconstituting a 5′ overhang; (c) an oligonucleotide of about 1 to about20 nucleotides covalently bonded to the 3′ end of oligoA andconstituting a 3′ overhang; (d) and an oligonucleotide of about 4 toabout 20 nucleomonomers covalently bonded to the 3′ end of oligoA andthe 5′ end of oligoB and constituting a loop structure, where thenucleomonomers are selected from the group consisting of G and A.

In one embodiment, the double-stranded duplex of an oligonucleotide ofthe invention is from between about 12 to about 50 nucleomonomers inlength, i.e., the number of nucleotides of the double-strandedoligonucleotide which hybridize to the complementary sequence of thedouble-stranded oligonucleotide to form the double-stranded duplexstructure is from about 12 to about 50 nucleomonomers in length. Inanother embodiment, the double-stranded duplex of an oligonucleotide ofthe invention is from between about 12 to about 40 nucleomonomers inlength.

In one embodiment, the double-stranded duplex of an oligonucleotide ofthe invention is at least about 25 nucleomonomers in length. In oneembodiment, the double-stranded duplex is greater than about 25nucleomonomers in length. In one embodiment, a double-stranded duplex isat least about 26, 27, 28, 29, 30, at least about 40, at least about 50,at least about 60, at least about 70, at least about 80, or at leastabout 90 nucleomonomers in length. In another embodiment, thedouble-stranded duplex is less than about 25 nucleomonomers in length.In one embodiment, a double-stranded duplex is at least about 10, atleast about 15, at least about 20, at least about 22, at least about 23or at least about 24 nucleomonomers in length.

In one embodiment, the number of Ns in each strand of the duplex isabout 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.In another embodiment, the number of Ns in each strand of the duplex isabout 30, 35, 40, 45, or 50. In one embodiment, the number of Ns in eachstrand of the duplex is about 19. In a preferred embodiment, the numberof Ns in each strand of the duplex is about 27. In another embodiment,the number of Ns in each strand of the duplex is about 27 (e.g., is 26,27, or 28). In another embodiment, the number of Ns in each strand ofthe duplex is 27.

In one embodiment, an individual nucleic acid molecule of adouble-stranded oligonucleotide of the invention is at least about 25nucleomonomers in length. For example, when the double-strandedoligonucleotide of the invention is comprised of one nucleic acidmolecule, that individual molecule is at least about 25 nucleomonomersin length or when the double-stranded oligonucleotide of the inventionis comprised of two separate nucleic acid molecules, the length of atleast one of the individual nucleic acid molecules is at least about 25nucleomonomers in length.

A variety of nucleotides of different lengths may be used. In oneembodiment, an individual nucleic acid molecule comprising adouble-stranded oligonucleotide of the invention is greater than about25 nucleomonomers in length. In one embodiment, an individual nucleicacid molecule comprising a double-stranded oligonucleotide of theinvention is at least about 26, 27, 28, 29, 30, at least about 40, atleast about 50, or at least about 60, at least about 70, at least about80, or at least about 90 nucleomonomers in length. In anotherembodiment, an individual nucleic acid molecule comprising adouble-stranded oligonucleotide of the invention is less than about 25nucleomonomers in length. In one embodiment, an individual nucleic acidmolecule comprising a double-stranded oligonucleotide of the inventionis at least about 10, at least about 15, at least about 20, at leastabout 22, at least about 23 or at least about 24 nucleomonomers inlength.

Double-stranded molecules of the invention may comprise a firstnucleotide sequence which is antisense to at least part of the targetgene and a second nucleotide sequence which is complementary to thefirst nucleotide sequence; i.e., is sense to at least part of the targetgene. In one embodiment, the second nucleotide sequence of thedouble-stranded molecule comprises a nucleotide sequence which is atleast about 100% complementary to the antisense molecule.

In another embodiment, the second nucleotide sequence of double-strandedmolecules of the invention may comprise a nucleotide sequence which isat least about 95% complementary to the antisense molecule. In anotherembodiment, the second nucleotide sequence of double-stranded moleculesof the invention may comprise a nucleotide sequence which is at leastabout 90% complementary to the antisense molecule. In anotherembodiment, the second nucleotide sequence of double-stranded moleculesof the invention may comprise a nucleotide sequence which is at leastabout 80% complementary to the antisense molecule. In anotherembodiment, the second nucleotide sequence of double-stranded moleculesof the invention may comprises a nucleotide sequence which is at leastabout 60% complementary to the antisense molecule. In anotherembodiment, the second nucleotide sequence of double-stranded moleculesof the invention may comprise a nucleotide sequence which is at leastabout 100% complementary to the antisense molecule.

To determine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-identical sequences can bedisregarded for comparison purposes). When a position in the firstsequence is occupied by the same nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The percent complementarity can be determined analogously; when aposition in one sequence occupied by a nucleotide that is complementaryto the nucleotide in the other sequence, then the molecules arecomplementary at that position.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twonucleotide sequences is determined using e.g., the GAP program in theGCG software package, using a NWSgapdna. CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two nucleotidesequences is determined using the algorithm of E. Meyers and W. Miller(Comput. Appl. Biosci. 4:11-17 (1988)) which has been incorporated intothe ALIGN program (version 2.0), using a PAM120 weight residue table, agap length penalty of 12 and a gap penalty of 4.

The nucleic acid sequences of the present invention can further be usedas a “query sequence” to perform alignments against sequences in publicdatabases. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. See, e.g.,the NIH website.

In yet another embodiment, a first antisense sequence of thedouble-stranded molecule hybridizes to its complementary second sequenceof the double-stranded molecule under stringent hybridizationconditions. As used herein, the term “hybridizes under stringentconditions” is intended to describe conditions for hybridization andwashing under which nucleotide sequences at least 60% complementary toeach other typically remain hybridized to each other. Preferably, theconditions are such that sequences at least about 70%, more preferablyat least about 80%, even more preferably at least about 85% or 90%complementary to each other typically remain hybridized to each other.

Such stringent conditions are known to those skilled in the art and canbe found in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., more preferably at60° C., and even more preferably at 65° C. Ranges intermediate to theabove-recited values, e.g., at 60-65° C. or at 55-60° C. are alsointended to be encompassed by the present invention. Alternatively,formamide can be included in the hybridization solution, using methodsand conditions also known in the art.

One of the sequences (or molecules) of the double-strandedoligonucleotide of the invention is antisense to the target gene. Asused herein, the term “antisense sequence” includes nucleotide sequenceswhich bind to the “sense” strand of the nucleotide sequence of thetarget gene (e.g., polynucleotides such as DNA, mRNA (includingpre-mRNA) molecules). When the antisense sequences of the invention bindto nucleic acid molecules, they can bind to any region of a nucleic acidmolecule, including e.g., introns, exons, 5′, or 3′ untranslatedregions. Antisense sequences that work by binding to a target andactivating RNase H preferably bind within an intron, an exon, the 5′untranslated region, or the 3′ untranslated region of a nucleic acidtarget molecule.

Preferably, the oligonucleotide compositions of the invention do notactivate the interferon pathway, e.g., as evidenced by the lack ofinduction of the double-stranded RNA, interferon-inducible proteinkinase, PKR.

In one embodiment, modifications are made to a double-stranded RNAmolecule which would normally activate the interferon pathway such thatthe interferon pathway is not activated. For example, the interferonpathway is activated by double-stranded unmodified RNA. The cellularrecognition of double-stranded RNA is highly specific and modifying oneor both of the strands of a double-stranded duplex enables thedouble-stranded RNA molecule to evade the double-stranded RNArecognition machinery of the cell but would still allow for theactivation of the RNAi pathway.

The ability of a double-stranded oligonucleotide to activate interferoncould be assessed by testing for expression of the double-stranded RNA,Interferon-Inducible Protein Kinase, PKR using techniques known in theart and also testing for the ability of the double-stranded molecule toeffect target gene inhibition. Accordingly, in one embodiment, theinvention provides a method of testing for the ability of adouble-stranded RNA molecule to induce interferon by testing for theability of the oligonucleotide to activate PKR. Compositions that do notactivate PKR (i.e., do not activate the interferon pathway) are thenselected for use to inhibit gene transcription in cells, e.g., intherapeutics or functional genomics.

Without being limited to any particular mechanism of action, anantisense sequence used in a double-stranded oligonucleotide compositionof the invention that can specifically hybridize with a nucleotidesequence within the target gene (i.e., can be complementary to anucleotide sequence within the target gene) may achieve its affectsbased on, e.g.,: (1) binding to target mRNA and sterically blocking theribosome complex from translating the mRNA; (2) binding to target mRNAand triggering mRNA cleavage by RNase H; (3) binding to double-strandedDNA in the nucleus and forming a triple helix; (4) hybridizing to openDNA loops created by RNA polymerase; (5) interfering with mRNA splicing;(6) interfering with transport of mRNA from the nucleus to thecytoplasm; or (7) interfering with translation through inhibition of thebinding of initiation factors or assembly of ribosomal subunits (i.e.,at the start codon).

In one embodiment, an antisense sequence of the double-strandedoligonucleotides of the invention is complementary to a target nucleicacid sequence over at least about 80% of the length of the antisensesequence. In another embodiment, the antisense sequence of thedouble-stranded oligonucleotide of the invention is complementary to atarget nucleic acid sequence over at least about 90-95% of the length ofthe antisense sequence. In another embodiment, the antisense sequence ofthe double-stranded oligonucleotide of the invention is complementary toa target nucleic acid sequence over the entire length of the antisensesequence.

In yet another embodiment, an antisense sequence of the double-strandedoligonucleotide hybridizes to at least a portion of the target geneunder stringent hybridization conditions.

In one embodiment, antisense sequences of the invention aresubstantially complementary to a target nucleic acid sequence. In oneembodiment, an antisense RNA molecule comprises a nucleotide sequencewhich is at least about 100% complementary to a portion of the targetgene. In another embodiment, an antisense RNA molecule comprises anucleotide sequence which is at least about 90% complementary to aportion of the target gene. In another embodiment, an antisense RNAmolecule comprises a nucleotide sequence which is at least about 80%complementary to a portion of the target gene. In another embodiment, anantisense RNA molecule comprises a nucleotide sequence which is at leastabout 60% complementary to a portion of the target gene. In anotherembodiment, an antisense RNA molecule comprises a nucleotide sequencewhich is at least about 100% complementary to a portion of the targetgene. Preferably, no loops greater than about 8 nucleotides are formedby areas of non-complementarity between the oligonucleotide and thetarget.

In one embodiment, an antisense nucleotide sequence of the invention iscomplementary to a target nucleic acid sequence over at least about 80%of the length of the antisense sequence. In another embodiment, anantisense sequence of the invention is complementary to a target nucleicacid sequence over at least about 90-95% of the length of the antisensesequence. In another embodiment, an antisense sequence of the inventionis complementary to a target nucleic acid sequence over the entirelength of the antisense sequence.

The antisense sequences used in an oligonucleotide composition of theinvention may be of any type, e.g., including morpholinooligonucleotides, RNase H activating oligonucleotides, or ribozymes.

In one embodiment, a double-stranded oligonucleotide of the inventioncan comprise (i.e., be a duplex of) one nucleic acid molecule which isDNA and one nucleic acid molecule which is RNA.

Antisense sequences of the invention can be “chimeric oligonucleotides”which comprise an RNA-like and a DNA-like region. The language “RNase Hactivating region” includes a region of an oligonucleotide, e.g., achimeric oligonucleotide, that is capable of recruiting RNase H tocleave the target RNA strand to which the oligonucleotide binds.Typically, the RNase activating region contains a minimal core (of atleast about 3-5, typically between about 3-12, more typically, betweenabout 5-12, and more preferably between about 5-10 contiguousnucleomonomers) of DNA or DNA-like nucleomonomers. (See, e.g., U.S. Pat.No. 5,849,902). Preferably, the RNase H activating region comprisesabout nine contiguous deoxyribose containing nucleomonomers.

In one embodiment, the contiguous nucleomonomers are linked by asubstitute linkage, e.g., a phosphorothioate linkage. In one embodiment,an antisense sequence of the invention is unstable, i.e., is degraded ina cell, in the absence of the second strand (or self complementarysequence) which forms a double-stranded oligonucleotide of theinvention. For example, in one embodiment, a chimeric antisense sequencecomprises unmodified DNA nucleomonomers in the gap rather thanphosphorothioate DNA.

The language “non-activating region” includes a region of an antisensesequence, e.g., a chimeric oligonucleotide, that does not recruit oractivate RNase H. Preferably, a non-activating region does not comprisephosphorothioate DNA. The oligonucleotides of the invention comprise atleast one non-activating region. In one embodiment, the non-activatingregion can be stabilized against nucleases or can provide specificityfor the target by being complementary to the target and forming hydrogenbonds with the target nucleic acid molecule, which is to be bound by theoligonucleotide.

Antisense sequences of the present invention may include “morpholinooligonucleotides.” Morpholino oligonucleotides are non-ionic andfunction by an RNase H-independent mechanism. Each of the 4 geneticbases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholinooligonucleotides is linked to a 6-membered morpholine ring. Morpholinooligonucleotides are made by joining the 4 different subunit types by,e.g., non-ionic phosphorodiamidate inter-subunit linkages. An example ofa 2 subunit morpholino oligonucleotide is shown in FIG. 13.

Morpholino oligonucleotides have many advantages including: completeresistance to nucleases (Antisense & Nucl. Acid Drug Dev. 1996. 6:267);predictable targeting (Biochemica Biophysica Acta. 1999. 1489:141);reliable activity in cells (Antisense & Nucl. Acid Drug Dev. 1997.7:63); excellent sequence specificity (Antisense & Nucl. Acid Drug Dev.1997. 7:151); minimal non-antisense activity (Biochemica BiophysicaActa. 1999. 1489:141); and simple osmotic or scrape delivery (Antisense& Nucl. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides arealso preferred because of their non-toxicity at high doses. A discussionof the preparation of morpholino oligonucleotides can be found inAntisense & Nucl. Acid Drug Dev. 1997. 7:187.

Uptake of Oligonucleotides by Cells

Oligonucleotides and oligonucleotide compositions are contacted with(i.e., brought into contact with, also referred to herein asadministered or delivered to) and taken up by one or more cells or acell lysate. The term “cells” includes prokaryotic and eukaryotic cells,preferably vertebrate cells, and, more preferably, mammalian cells. In apreferred embodiment, the oligonucleotide compositions of the inventionare contacted with human cells.

Oligonucleotide compositions of the invention can be contacted withcells in vitro, e.g., in a test tube or culture dish, (and may or maynot be introduced into a subject) or in vivo, e.g., in a subject such asa mammalian subject. Oligonucleotides are taken up by cells at a slowrate by endocytosis, but endocytosed oligonucleotides are generallysequestered and not available, e.g., for hybridization to a targetnucleic acid molecule. In one embodiment, cellular uptake can befacilitated by electroporation or calcium phosphate precipitation.However, these procedures are only useful for in vitro or ex vivoembodiments, are not convenient and, in some cases, are associated withcell toxicity.

In another embodiment, delivery of oligonucleotides into cells can beenhanced by suitable art recognized methods including calcium phosphate,DMSO, glycerol or dextran, electroporation, or by transfection, e.g.,using cationic, anionic, or neutral lipid compositions or liposomesusing methods known in the art (see e.g., WO 90/14074; WO 91/16024; WO91/17424; U.S. Pat. No. 4,897,355; Bergan et al. 1993. Nucleic AcidsResearch. 21:3567). Enhanced delivery of oligonucleotides can also bemediated by the use of vectors (See e.g., Shi, Y. 2003. Trends Genet2003 Jan. 19: 9; Reichhart J M et al. Genesis. 2002. 34(1-2):160-4, Yuet al. 2002. Proc. Natl. Acad Sci. USA 99:6047; Sui et al. 2002. Proc.Natl. Acad Sci. USA 99:5515) viruses, polyamine or polycation conjugatesusing compounds such as polylysine, protamine, or N1,N12-bis(ethyl)spermine (see, e.g., Bartzatt, R. et al. 1989. Biotechnol.Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl. Acad. Sci.88:4255).

The optimal protocol for uptake of oligonucleotides will depend upon anumber of factors, the most crucial being the type of cells that arebeing used. Other factors that are important in uptake include, but arenot limited to, the nature and concentration of the oligonucleotide, theconfluence of the cells, the type of culture the cells are in (e.g., asuspension culture or plated) and the type of media in which the cellsare grown. Examples of different protocols for different cell types areset forth in the Examples section.

Employing a 1 milliliter final volume solely as a reference volume,exemplary amounts of reagents which may be used to transfect cells withnucleic acid molecules of the invention (e.g., double-strandedoligonucleotides such as, for example, STEALTH™ RNA) include thefollowing. For nucleic acid molecules of the invention (e.g., STEALTH™RNA), the amount present may be between about 0.1 picomoles and about900 nanomoles, between about 0.1 picomoles and about 700 nanomoles,between about 0.1 picomoles and about 500 nanomoles, between about 0.1picomoles and about 300 nanomoles, between about 0.1 picomoles and about200 nanomoles, between about 0.1 picomoles and about 100 nanomoles,between about 0.1 picomoles and about 50 nanomoles, between about 0.1picomoles and about 25 nanomoles, between about 0.1 picomoles and about1.0 nanomole, between about 0.1 picomoles and about 800 picomoles,between about 0.1 picomoles and about 600 picomoles, between about 0.1picomoles and about 500 picomoles, between about 0.1 picomoles and about300 picomoles, between about 0.1 picomoles and about 200 picomoles,between about 1 picomole and about 900 nanomoles, between about 1 andabout 600 picomoles, between about 1 and about 500 picomoles, betweenabout 1 and about 400 picomoles, between about 100 and about 800picomoles, between about 200 and about 800 picomoles, between about 300and about 800 picomoles, between about 400 and about 800 picomoles,between about 200 and about 700 picomoles, between about 50 and about800 picomoles, between about 50 and about 500 picomoles, between about50 and about 400 picomoles, between about 50 and about 300 picomoles,between about 50 and about 200 picomoles, between about 100 and about200 picomoles, between about 100 and about 300 picomoles, between about1 nanomole and about 800 nanomoles, between about 100 nanomoles andabout 800 nanomoles, between about 100 nanomoles and about 900nanomoles, between about 200 nanomoles and about 900 nanomoles, betweenabout 300 nanomoles and about 900 nanomoles, between about 400 nanomolesand about 900 nanomoles, or between about 500 nanomoles and about 900nanomoles.

Further, the total number of cells present may be between about 1.0×10³and about 1.0×10⁶, between about 4.0×10³ and about 1.0×10⁶, betweenabout 5.0×10³ and about 1.0×10⁶, between about 8.0×10³ and about1.0×10⁶, between about 9.0×10³ and about 5.0×10⁵, between about 1.0×10³and about 1.0×10⁵, between about 5.0×10⁴ and about 1.0×10⁴, betweenabout 4.0×10³ and about 5.0×10⁵, or between about 1.0×10⁴ and about1.0×10⁵.

The amount of LIPOFECTAMINE™ 2000 or OLIGOFECTAMINE™, when present as atransfection reagent may be between 0.5 nanoliters and 100 microliters,between 5 nanoliters and 100 microliters, between 50 nanoliters and 100microliters, between 100 nanoliters and 100 microliters, between 200nanoliters and 100 microliters, between 300 nanoliters and 100microliters, between 500 nanoliters and 100 microliters, between 750nanoliters and 100 microliters, between 1.0 microliter and 100microliters, between 10 microliters and 100 microliters, between 50microliters and 100 microliters, between 1.0 microliter and 75microliters, between 1.0 microliter and 50 microliters, or between 1.0microliters and 30 microliters.

Detectably labeled oligonucleotide controls may be contacted with cellsin concentrations between about 0.1 nanomoles and 1000 nanomoles,between about 1.0 nanomole and 1000 nanomoles, between about 5.0nanomoles and 1000 nanomoles, between about 10 nanomoles and 1000nanomoles, between about 20 nanomoles and 1000 nanomoles, between about40 nanomoles and 1000 nanomoles, between about 60 nanomoles and 1000nanomoles, between about 100 nanomoles and 1000 nanomoles, between about0.1 nanomole and 800 nanomoles, between about 0.1 nanomoles and 700nanomoles, between about 0.1 nanomoles and 600 nanomoles, between about0.1 nanomoles and 500 nanomoles, between about 0.1 nanomoles and 400nanomoles, between about 10 nanomoles and 600 nanomoles, between about10 nanomoles and 500 nanomoles, between about 10 nanomoles and 300nanomoles, between about 10 nanomoles and 200 nanomoles, between about10 nanomoles and 100 nanomoles, between about 10 nanomoles and 50nanomoles, or between about 20 nanomoles and 200 nanomoles.

Exemplary formulations of the above components are set out in theproduct literature and table set forth in Example 15.

Conjugating Agents

Conjugating agents bind to the oligonucleotide in a covalent manner. Inone embodiment, oligonucleotides can be derivatized or chemicallymodified by binding to a conjugating agent to facilitate cellularuptake. For example, covalent linkage of a cholesterol moiety to anoligonucleotide can improve cellular uptake by 5- to 10-fold which inturn improves DNA binding by about 10-fold (Boutorin et al., 1989, FEBSLetters 254:129-132). Conjugation of octyl, dodecyl, and octadecylresidues enhances cellular uptake by 3-, 4-, and 10-fold as compared tounmodified oligonucleotides (Vlassov et al., 1994, Biochimica etBiophysica Acta 1197:95-108). Similarly, derivatization ofoligonucleotides with poly-L-lysine can aid oligonucleotide uptake bycells (Schell, 1974, Biochem. Biophys. Acta 340:323, and Lemaitre etal., 1987, Proc. Natl. Acad. Sci. USA 84:648).

Certain protein carriers can also facilitate cellular uptake ofoligonucleotides, including, for example, serum albumin, nuclearproteins possessing signals for transport to the nucleus, and viral orbacterial proteins capable of cell membrane penetration. Therefore,protein carriers are useful when associated with or linked to theoligonucleotides. Accordingly, the present invention provides forderivatization of oligonucleotides with groups capable of facilitatingcellular uptake, including hydrocarbons and non-polar groups,cholesterol, long chain alcohols (i.e., hexanol), poly-L-lysine andproteins, as well as other aryl or steroid groups and polycations havinganalogous beneficial effects, such as phenyl or naphthyl groups,quinoline, anthracene or phenanthracene groups, fatty acids, fattyalcohols and sesquiterpenes, diterpenes, and steroids. A major advantageof using conjugating agents is to increase the initial membraneinteraction that leads to a greater cellular accumulation ofoligonucleotides.

Encapsulating Agents

Encapsulating agents entrap oligonucleotides within vesicles. In anotherembodiment of the invention, an oligonucleotide may be associated with acarrier or vehicle, e.g., liposomes or micelles, although other carrierscould be used, as would be appreciated by one skilled in the art.Liposomes are vesicles made of a lipid bilayer having a structuresimilar to biological membranes. Such carriers are used to facilitatethe cellular uptake or targeting of the oligonucleotide, or improve theoligonucleotide's pharmacokinetic or toxicologic properties.

For example, the oligonucleotides of the present invention may also beadministered encapsulated in liposomes, pharmaceutical compositionswherein the active ingredient is contained either dispersed or variouslypresent in corpuscles consisting of aqueous concentric layers adherentto lipidic layers. The oligonucleotides, depending upon solubility, maybe present both in the aqueous layer and in the lipidic layer, or inwhat is generally termed a liposomic suspension. The hydrophobic layer,generally but not exclusively, comprises phopholipids such as lecithinand sphingomyelin, steroids such as cholesterol, more or less ionicsurfactants such as diacetylphosphate, stearylamine, or phosphatidicacid, or other materials of a hydrophobic nature. The diameters of theliposomes generally range from about 15 nm to about 5 microns.

The use of liposomes as drug delivery vehicles offers severaladvantages. Liposomes increase intracellular stability, increase uptakeefficiency and improve biological activity. Liposomes are hollowspherical vesicles composed of lipids arranged in a similar fashion asthose lipids which make up the cell membrane. They have an internalaqueous space for entrapping water soluble compounds and range in sizefrom 0.05 to several microns in diameter. Several studies have shownthat liposomes can deliver nucleic acids to cells and that the nucleicacids remain biologically active. For example, a liposome deliveryvehicle originally designed as a research tool, such as Lipofectin orLIPOFECTAMINE™ 2000, can deliver intact nucleic acid molecules to cells.

Specific advantages of using liposomes include the following: they arenon-toxic and biodegradable in composition; they display longcirculation half-lives; and recognition molecules can be readilyattached to their surface for targeting to tissues. Finally,cost-effective manufacture of liposome-based pharmaceuticals, either ina liquid suspension or lyophilized product, has demonstrated theviability of this technology as an acceptable drug delivery system.

Complexing Agents

Complexing agents bind to the oligonucleotides of the invention by astrong but non-covalent attraction (e.g., an electrostatic, van derWaals, pi-stacking, etc. interaction). In one embodiment,oligonucleotides of the invention can be complexed with a complexingagent to increase cellular uptake of oligonucleotides. An example of acomplexing agent includes cationic lipids. Cationic lipids can be usedto deliver oligonucleotides to cells.

The term “cationic lipid” includes lipids and synthetic lipids havingboth polar and non-polar domains and which are capable of beingpositively charged at or around physiological pH and which bind topolyanions, such as nucleic acids, and facilitate the delivery ofnucleic acids into cells. In general cationic lipids include saturatedand unsaturated alkyl and alicyclic ethers and esters of amines, amides,or derivatives thereof. Straight-chain and branched alkyl and alkenylgroups of cationic lipids can contain, e.g., from 1 to about 25 carbonatoms. Preferred straight chain or branched alkyl or alkene groups havesix or more carbon atoms. Alicyclic groups include cholesterol and othersteroid groups. Cationic lipids can be prepared with a variety ofcounterions (anions) including, e.g., Cl⁻, Br⁻, I⁻, F⁻, acetate,trifluoroacetate, sulfate, nitrite, and nitrate.

Examples of cationic lipids include polyethylenimine, polyamidoamine(PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA andDOPE), Lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE,Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL,San Luis Obispo, Calif.). Exemplary cationic liposomes can be made fromN-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA),N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate(DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol(DC-Chol),2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; anddimethyldioctadecylammonium bromide (DDAB). The cationic lipidN-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),for example, was found to increase 1000-fold the antisense effect of aphosphothioate oligonucleotide. (Vlassov et al., 1994, Biochimica etBiophysica Acta 1197:95-108). Oligonucleotides can also be complexedwith, e.g., poly(L-lysine) or avidin and lipids may, or may not, beincluded in this mixture, e.g., steryl-poly(L-lysine).

Cationic lipids have been used in the art to deliver oligonucleotides tocells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430;5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA93:3176; Hope et al. 1998. Molecular Membrane Biology 15:1). Other lipidcompositions which can be used to facilitate uptake of the instantoligonucleotides can be used in connection with the claimed methods. Inaddition to those listed supra, other lipid compositions are also knownin the art and include, e.g., those taught in U.S. Pat. No. 4,235,871;U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.

In one embodiment lipid compositions can further comprise agents, e.g.,viral proteins to enhance lipid-mediated transfections ofoligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536). Inanother embodiment, oligonucleotides are contacted with cells as part ofa composition comprising an oligonucleotide, a peptide, and a lipid astaught, e.g., in U.S. Pat. No. 5,736,392. Improved lipids have also beendescribed which are serum resistant (Lewis, et al., 1996. Proc. Natl.Acad. Sci. 93:3176). Cationic lipids and other complexing agents act toincrease the number of oligonucleotides carried into the cell throughendocytosis.

In another embodiment N-substituted glycine oligonucleotides (peptoids)can be used to optimize uptake of oligonucleotides. Peptoids have beenused to create cationic lipid-like compounds for transfection (Murphy,et al., 1998. Proc. Natl. Acad. Sci. 95:1517). Peptoids can besynthesized using standard methods (e.g., Zuckermann, R. N., et al.1992. J. Am. Chem. Soc. 114:10646; Zuckermann, R. N., et al. 1992. Int.J. Peptide Protein Res. 40:497). Combinations of cationic lipids andpeptoids, liptoids, can also be used to optimize uptake of the subjectoligonucleotides (Hunag, et al., 1998. Chemistry and Biology. 5:345).Liptoids can be synthesized by elaborating peptoid oligonucleotides andcoupling the amino terminal submonomer to a lipid via its amino group(Hunag, et al., 1998. Chemistry and Biology. 5:345).

It is known in the art that positively charged amino acids can be usedfor creating highly active cationic lipids (Lewis et al. 1996. Proc.Natl. Acad. Sci. U.S.A. 93:3176). In one embodiment, a composition fordelivering oligonucleotides of the invention comprises a number ofarginine, lysine, histidine or ornithine residues linked to a lipophilicmoiety (see e.g., U.S. Pat. No. 5,777,153).

In another embodiment, a composition for delivering oligonucleotides ofthe invention comprises a peptide having from between about one to aboutfour basic residues. These basic residues can be located, e.g., on theamino terminal, C-terminal, or internal region of the peptide. Familiesof amino acid residues having similar side chains have been defined inthe art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine (canalso be considered non-polar), asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Apart from the basic amino acids, a majority or all of theother residues of the peptide can be selected from the non-basic aminoacids, e.g., amino acids other than lysine, arginine, or histidine.Preferably a preponderance of neutral amino acids with long neutral sidechains are used. For example, a peptide such as (N-term)His-Ile-Trp-Leu-Ile-Tyr-Leu-Trp-Ile-Val-(C-term) (SEQ ID NO: 14) couldbe used. In one embodiment such a composition can be mixed with thefusogenic lipid DOPE as is well known in the art.

In one embodiment, the cells to be contacted with an oligonucleotidecomposition of the invention are contacted with a mixture comprising theoligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 12 hoursto about 24 hours. In another embodiment, the cells to be contacted withan oligonucleotide composition are contacted with a mixture comprisingthe oligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 1 andabout five days. In one embodiment, the cells are contacted with amixture comprising a lipid and the oligonucleotide for between aboutthree days to as long as about 30 days. In another embodiment, a mixturecomprising a lipid is left in contact with the cells for at least aboutfive to about 20 days. In another embodiment, a mixture comprising alipid is left in contact with the cells for at least about seven toabout 15 days.

For example, in one embodiment, an oligonucleotide composition can becontacted with cells in the presence of a lipid such as cytofectin CS orGSV (available from Glen Research; Sterling, Va.), GS3815, GS2888 forprolonged incubation periods as described herein.

In one embodiment the incubation of the cells with the mixturecomprising a lipid and an oligonucleotide composition does not reducethe viability of the cells. Preferably, after the transfection periodthe cells are substantially viable. In one embodiment, aftertransfection, the cells are between at least about 70% and at leastabout 100% viable. In another embodiment, the cells are between at leastabout 80% and at least about 95% viable. In yet another embodiment, thecells are between at least about 85% and at least about 90% viable.

In one embodiment, oligonucleotides are modified by attaching a peptidesequence that transports the oligonucleotide into a cell, referred toherein as a “transporting peptide.” In one embodiment, the compositionincludes an oligonucleotide which is complementary to a target nucleicacid molecule encoding the protein, and a covalently attachedtransporting peptide.

The language “transporting peptide” includes an amino acid sequence thatfacilitates the transport of an oligonucleotide into a cell. Exemplarypeptides which facilitate the transport of the moieties to which theyare linked into cells are known in the art, and include, e.g., HIV TATtranscription factor, lactoferrin, Herpes VP22 protein, and fibroblastgrowth factor 2 (Pooga et al. 1998. Nature Biotechnology. 16:857; andDerossi et al. 1998. Trends in Cell Biology. 8:84; Elliott and O'Hare.1997. Cell 88:223).

For example, in one embodiment, the transporting peptide comprises anamino acid sequence derived from the antennapedia protein. Preferably,the peptide comprises amino acids 43-58 of the antennapedia protein(Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys) (SEQID NO: 15) or a portion or variant thereof that facilitates transport ofan oligonucleotide into a cell (see, e.g., WO 91/1898; Derossi et al.1998. Trends Cell Biol. 8:84). Exemplary variants are shown in Derossiet al., supra.

In one embodiment, the transporting peptide comprises an amino acidsequence derived from the transportan, galanin (1-12)-Lys-mastoparan(1-14) amide, protein. (Pooga et al. 1998. Nature Biotechnology 16:857).Preferably, the peptide comprises the amino acids of the transportanprotein shown in the sequence GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:16) or a portion or variant thereof that facilitates transport of anoligonucleotide into a cell.

In one embodiment, the transporting peptide comprises an amino acidsequence derived from the HIV TAT protein. Preferably, the peptidecomprises amino acids 37-72 of the HIV TAT protein, e.g., shown in thesequence C(Acm)FITKALGISYGRKKRRQRRRPPQC (SEQ ID NO: 17) (TAT 37-60;where C(Acm) is Cys-acetamidomethyl) or a portion or variant thereof,e.g., C(Acm)GRKKRRQRRRPPQC (SEQ ID NO: 18) (TAT 48-40) orC(Acm)LGISYGRKKRRQRRPPQC (SEQ ID NO: 19) (TAT 43-60) that facilitatestransport of an oligonucleotide into a cell (Vives et al. 1997. J. Biol.Chem. 272:16010). In another embodiment the peptide(G)CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ (SEQ ID NO: 20) can be used.

Portions or variants of transporting peptides can be readily tested todetermine whether they are equivalent to these peptide portions bycomparing their activity to the activity of the native peptide, e.g.,their ability to transport fluorescently-labeled oligonucleotides tocells. Fragments or variants that retain the ability of the nativetransporting peptide to transport an oligonucleotide into a cell arefunctionally equivalent and can be substituted for the native peptides.

Oligonucleotides can be attached to the transporting peptide using knowntechniques, e.g., (Prochiantz, A. 1996. Curr. Opin. Neurobiol. 6:629;Derossi et al. 1998. Trends Cell Biol. 8:84; Troy et al. 1996. J.Neurosci. 16:253), Vives et al. 1997. J. Biol. Chem. 272:16010). Forexample, in one embodiment, oligonucleotides bearing an activated thiolgroup are linked via that thiol group to a cysteine present in atransport peptide (e.g., to the cysteine present in the β turn betweenthe second and the third helix of the antennapedia homeodomain astaught, e.g., in Derossi et al. 1998. Trends Cell Biol. 8:84;Prochiantz. 1996. Current Opinion in Neurobiol. 6:629; Allinquant et al.1995. J. Cell Biol. 128:919). In another embodiment, a Boc-Cys-(Npys)OHgroup can be coupled to the transport peptide as the last (N-terminal)amino acid and an oligonucleotide bearing an SH group can be coupled tothe peptide (Troy et al. 1996. J. Neurosci. 16:253).

In one embodiment, a linking group can be attached to a nucleomonomerand the transporting peptide can be covalently attached to the linker.In one embodiment, a linker can function as both an attachment site fora transporting peptide and can provide stability against nucleases.Examples of suitable linkers include substituted or unsubstituted C₁-C₂₀alkyl chains, C₂-C₂₀ alkenyl chains, C₂-C₂₀ alkynyl chains, peptides,and heteroatoms (e.g., S, O, NH, etc.). Other exemplary linkers includebifunctional crosslinking agents such assulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smithet al. Biochem J 1991. 276: 417-2).

In one embodiment, oligonucleotides of the invention are synthesized asmolecular conjugates which utilize receptor-mediated endocytoticmechanisms for delivering genes into cells (see, e.g., Bunnell et al.1992. Somatic Cell and Molecular Genetics. 18:559, and the referencescited therein).

Targeting Agents

The delivery of oligonucleotides can also be improved by targeting theoligonucleotides to a cellular receptor. The targeting moieties can beconjugated to the oligonucleotides or attached to a carrier group (i.e.,poly(L-lysine) or liposomes) linked to the oligonucleotides. This methodis well suited to cells that display specific receptor-mediatedendocytosis.

For instance, oligonucleotide conjugates to 6-phosphomannosylatedproteins are internalized 20-fold more efficiently by cells expressingmannose 6-phosphate specific receptors than free oligonucleotides. Theoligonucleotides may also be coupled to a ligand for a cellular receptorusing a biodegradable linker. In another example, the delivery constructis mannosylated streptavidin which forms a tight complex withbiotinylated oligonucleotides. Mannosylated streptavidin was found toincrease 20-fold the internalization of biotinylated oligonucleotides.(Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

In addition specific ligands can be conjugated to the polylysinecomponent of polylysine-based delivery systems. For example,transferrin-polylysine, adenovirus-polylysine, and influenza virushemagglutinin HA-2 N-terminal fusogenic peptides-polylysine conjugatesgreatly enhance receptor-mediated DNA delivery in eucaryotic cells.Mannosylated glycoprotein conjugated to poly(L-lysine) in aveolarmacrophages has been employed to enhance the cellular uptake ofoligonucleotides. Liang et al. 1999. Pharmazie 54:559-566.

Because malignant cells have an increased need for essential nutrientssuch as folic acid and transferrin, these nutrients can be used totarget oligonucleotides to cancerous cells. For example, when folic acidis linked to poly(L-lysine) enhanced oligonucleotide uptake is seen inpromyelocytic leukaemia (HL-60) cells and human melanoma (M-14) cells.Ginobbi et al. 1997. Anticancer Res. 17:29. In another example,liposomes coated with maleylated bovine serum albumin, folic acid, orferric protoporphyrin IX, show enhanced cellular uptake ofoligonucleotides in murine macrophages, KB cells, and 2.2.15 humanhepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.

Liposomes naturally accumulate in the liver, spleen, andreticuloendothelial system (so-called, passive targeting). By couplingliposomes to various ligands such as antibodies are protein A, they canbe actively targeted to specific cell populations. For example, proteinA-bearing liposomes may be pretreated with H-2K specific antibodieswhich are targeted to the mouse major histocompatibility complex-encodedH-2K protein expressed on L cells. (Vlassov et al. 1994. Biochimica etBiophysica Acta 1197:95-108).

Assays of Oligonucleotide Stability

Preferably, the double-stranded oligonucleotides of the invention arestabilized, i.e., substantially resistant to endonuclease andexonuclease degradation. An oligonucleotide is defined as beingsubstantially resistant to nucleases when it is at least about 3-foldmore resistant to attack by an endogenous cellular nuclease, and ishighly nuclease resistant when it is at least about 6-fold moreresistant than a corresponding, single-stranded oligonucleotide. Thiscan be demonstrated by showing that the oligonucleotides of theinvention are substantially resistant to nucleases using techniqueswhich are known in the art.

One way in which substantial stability can be demonstrated is by showingthat the oligonucleotides of the invention function when delivered to acell, e.g., that they reduce transcription or translation of targetnucleic acid molecules, e.g., by measuring protein levels or bymeasuring cleavage of mRNA. Assays which measure the stability of targetRNA can be performed at about 24 hours post-transfection (e.g., usingNorthern blot techniques, RNase Protection Assays, or QC-PCR assays asknown in the art). Alternatively, levels of the target protein can bemeasured. Preferably, in addition to testing the RNA or protein levelsof interest, the RNA or protein levels of a control, non-targeted genewill be measured (e.g., actin, or preferably a control with sequencesimilarity to the target) as a specificity control. RNA or proteinmeasurements can be made using any art-recognized technique. Preferably,measurements will be made beginning at about 16-24 hours posttransfection. (M. Y. Chiang, et al. 1991. J Biol Chem. 266:18162-71; T.Fisher, et al. 1993. Nucleic Acids Research. 21 3857).

The ability of an oligonucleotide composition of the invention toinhibit protein synthesis can be measured using techniques which areknown in the art, for example, by detecting an inhibition in genetranscription or protein synthesis. For example, Nuclease S1 mapping canbe performed. In another example, Northern blot analysis can be used tomeasure the presence of RNA encoding a particular protein. For example,total RNA can be prepared over a cesium chloride cushion (see, e.g.,Ausebel et al., 1987. Current Protocols in Molecular Biology (Greene &Wiley, New York)). Northern blots can then be made using the RNA andprobed (see, e.g., Id.). In another example, the level of the specificmRNA produced by the target protein can be measured, e.g., using PCR. Inyet another example, Western blots can be used to measure the amount oftarget protein present. In still another embodiment, a phenotypeinfluenced by the amount of the protein can be detected. Techniques forperforming Western blots are well known in the art, see, e.g., Chen etal. J. Biol. Chem. 271:28259.

In another example, the promoter sequence of a target gene can be linkedto a reporter gene and reporter gene transcription (e.g., as describedin more detail below) can be monitored. Alternatively, oligonucleotidecompositions that do not target a promoter can be identified by fusing aportion of the target nucleic acid molecule with a reporter gene so thatthe reporter gene is transcribed. By monitoring a change in theexpression of the reporter gene in the presence of the oligonucleotidecomposition, it is possible to determine the effectiveness of theoligonucleotide composition in inhibiting the expression of the reportergene. For example, in one embodiment, an effective oligonucleotidecomposition will reduce the expression of the reporter gene.

A “reporter gene” is a nucleic acid that expresses a detectable geneproduct, which may be RNA or protein. Detection of mRNA expression maybe accomplished by Northern blotting and detection of protein may beaccomplished by staining with antibodies specific to the protein.Preferred reporter genes produce a readily detectable product. Areporter gene may be operably linked with a regulatory DNA sequence suchthat detection of the reporter gene product provides a measure of thetranscriptional activity of the regulatory sequence. In preferredembodiments, the gene product of the reporter gene is detected by anintrinsic activity associated with that product. For instance, thereporter gene may encode a gene product that, by enzymatic activity,gives rise to a detectable signal based on color, fluorescence, orluminescence. Examples of reporter genes include, but are not limitedto, those coding for chloramphenicol acetyl transferase (CAT),luciferase, β-galactosidase, and alkaline phosphatase.

One skilled in the art would readily recognize numerous reporter genessuitable for use in the present invention. These include, but are notlimited to, chloramphenicol acetyltransferase (CAT), luciferase, humangrowth hormone (hGH), and beta-galactosidase. Examples of such reportergenes can be found in F. A. Ausubel et al., Eds., Current Protocols inMolecular Biology, John Wiley & Sons, New York, (1989). Any gene thatencodes a detectable product, e.g., any product having detectableenzymatic activity or against which a specific antibody can be raised,can be used as a reporter gene in the present methods.

One reporter gene system is the firefly luciferase reporter system.(Gould, S. J., and Subramani, S. 1988. Anal. Biochem., 7:404-408incorporated herein by reference). The luciferase assay is fast andsensitive. In this assay, a lysate of the test cell is prepared andcombined with ATP and the substrate luciferin. The encoded enzymeluciferase catalyzes a rapid, ATP dependent oxidation of the substrateto generate a light-emitting product. The total light output is measuredand is proportional to the amount of luciferase present over a widerange of enzyme concentrations.

CAT is another frequently used reporter gene system; a major advantageof this system is that it has been an extensively validated and iswidely accepted as a measure of promoter activity. (Gorman C. M.,Moffat, L. F., and Howard, B. H. 1982. Mol. Cell. Biol., 2:1044-1051).In this system, test cells are transfected with CAT expression vectorsand incubated with the candidate substance within 2-3 days of theinitial transfection. Thereafter, cell extracts are prepared. Theextracts are incubated with acetyl CoA and radioactive chloramphenicol.Following the incubation, acetylated chloramphenicol is separated fromnonacetylated form by thin layer chromatography. In this assay, thedegree of acetylation reflects the CAT gene activity with the particularpromoter.

Another suitable reporter gene system is based on immunologic detectionof hGH. This system is also quick and easy to use. (Selden, R.,Burke-Howie, K. Rowe, M. E., Goodman, H. M., and Moore, D. D. (1986),Mol. Cell, Biol., 6:3173-3179 incorporated herein by reference). The hGHsystem is advantageous in that the expressed hGH polypeptide is assayedin the media, rather than in a cell extract. Thus, this system does notrequire the destruction of the test cells. It will be appreciated thatthe principle of this reporter gene system is not limited to hGH butrather adapted for use with any polypeptide for which an antibody ofacceptable specificity is available or can be prepared.

In one embodiment, nuclease stability of a double-strandedoligonucleotide of the invention is measured and compared to a control,e.g., an RNAi molecule typically used in the art (e.g., a duplexoligonucleotide of less than 25 nucleotides in length and comprising 2nucleotide base overhangs) or an unmodified RNA duplex with blunt ends.

Monitoring the Effects of Oligonucleotide

Monitoring the effects of double-stranded oligonucleotides of theinvention on cell lines can by performed by the addition of staincompounds to cells, tissues, or organisms undergoing experiments ortreatment. Addition of a stain compound to cells, tissues, or organismsin an experiment allows for the monitoring of the effects of theoligonucleotide at one or more discrete time points or in real time withcontinuous monitoring. Effects monitored can include apoptosis, cellularhealth and vitality, cell proliferation, cellular phenotypic changes,and so on. Stain compounds can be fluorescent or otherwise cause adetectable signal when interacting with a target.

Methods for monitoring the effects of an oligonucleotide of the presentinvention generally comprise contacting one or more cells with anoligonucleotide molecule and a stain compound, and detecting a signalfrom the cells. The contacting step can occur in one step, where theoligonucleotide molecule and the stain compound are introduced into thecell simultaneously. Alternatively, the contacting step can be performedstepwise, where the stain molecule is introduced into the cell, and thenthe oligonucleotide molecule is introduced into the cell, or vise versa.The contacting step can include the addition of cellular uptake agentssuch as a surfactant. Multiple oligonucleotide molecules can be used,such as 2, 3, 4, 5, 6, and so on. Multiple stain compounds can be used,such as 2, 3, 4, 5, 6, and so on.

Stain compounds can generally be any compound that generates adetectable signal upon interaction with a target. Compounds typicallyare luminescent (e.g. fluorescent, chemiluminescent, or phosphorescent).Stain compounds can generate the detectable signal directly (i.e. signalis generated upon interaction), or indirectly by including a thirdcompound (e.g. the stain compound can be an enzyme that acts upon asubstrate that becomes fluorescent). Examples of stain compounds includeDNA labeled with a fluorescent molecule, RNA labeled with a fluorescentmolecule, an antibody labeled with a fluorescent molecule, a Fabfragment labeled with a fluorescent molecule, and so on. The fluorescentmolecule can be an organic compound, or a protein such as greenfluorescent protein (GFP). Antibodies can be a labeled primary antibody,or a combination of an unlabeled primary antibody and a labeledsecondary antibody. Antibodies can be labeled with a fluorescent orother detectable group, or can be labeled with an enzyme (such as aperoxidase, alkaline phosphatase, galactosidase, luciferase, orlactamase) that can react with a substrate. Stain compounds can interactwith various targets. Targets include nucleic acid (e.g. DNA), proteins,peptides, and lipids. Targets can also include cellular structures suchas cytoplasm, cytoskeleton, endoplasmic reticulum (ER), golgi,lysosomes, mitochondria, nucleus, nucleoli, peroxisomes, and plasmamembrane.

Specific examples of stain compounds useful for studying changes in cellstructure include 4′,6-diamidino-2-phenylindole dihydrochloride (usefulcounterstain for nucleus and chromosomes), Hoechst 33342trihydrochloride trihydrate (useful cell-permeant nuclear counterstainthat emits blue fluorescence when bound to dsDNA; can be used todistinguish condensed pycnotic nuclei in apoptotic cells and forcell-cycle studies with BrdU), SYTOX Blue (a blue-fluorescent nuclearand chromosome counterstain that is impermeant to live cells), SYTOXGreen (a green-fluorescent nuclear and chromosome counterstain that isimpermeant to live cells), YO-PRO-1 iodide (a carbocyanine nucleic acidstain useful for identifying apoptotic cells), BO-PRO-1 iodide (acarbocyanine nucleic acid stain), SYTO 59 (a cell-permeant nucleic acidstain), and TO-PRO-3 iodide (a carbocyanine monomer useful as a deadcell indicator).

Specific examples of stain compounds useful for studying DNAfragmentation include 5-bromo-2′-deoxyuridine 5′-triphosphate (BrdUTP)with Alexa Fluor 488 anti-BrdU, anti-bromodeoxyuridine, mouse IgG1,monoclonal PRB-1 Alexa Fluor 488 conjugate (anti-BrdU, Alexa Fluor 488conjugate), and anti-bromodeoxyuridine, mouse IgG1, monoclonal PRB-1Alexa Fluor 594 conjugate (anti-BrdU, Alexa Fluor 594 conjugate).

Specific examples of stain compounds useful for studying cellproliferation include CyQUANT, and carboxyfluorescein diacetatesuccinimidyl ester.

Specific examples of stain compounds useful for studying apoptosisinclude caspase substrates rhodamine 110,bis-(N-CBZ-L-isoleucyl-L-glutamyl-L-threonyl-L-aspartic acid amide),Z-DEVD-AMC (C₃₆H₄₁N₅O₁₄), Z-DEVD-AMC (C₃₆H₄₁N₅O₁₄), and rhodamine 110,bis-(L-aspartic acid amide). Compounds useful for studyingphosphatidylserine exposure include recombinant annexin V conjugated togreen-fluorescent Alexa Fluor 488 dye, green-fluorescent Alexa Fluor 488annexin with red-fluorescent propidium iodide nucleic acid stain, AlexaFluor 488 annexin V conjugate with SYTOX Green nucleic acid stain andC₁₂-resazurin, recombinant annexin V conjugated to allophycocyanin (APC)and a SYTOX Green nucleic acid stain, recombinant annexin V conjugatedto R-phycoerythrin (R-PE) and a SYTOX Green nucleic acid stain, AlexaFluor 568 annexin V conjugate, Alexa Fluor 594 annexin V conjugate,Alexa Fluor 350 annexin V conjugate, and Alexa Fluor 647 annexin Vconjugate.

Specific examples of stain compounds useful for studying changes inmitochondria include MitoTracker Red CMXRos (a red-fluorescent dye thatstains mitochondria in live cells), MitoTracker Green FM (agreen-fluorescent mitochondrial stain that stains live cells), andMitoTracker Orange CMTMRos (an orange-fluorescent mitochondrial stainthat stains live cells).

Specific examples of stain compounds useful for studying changes inlysosomes include LysoTracker Red DND-99 (a red-fluorescent dye thatstains acidic compartments in live cells), LysoTracker Green DND-26 (agreen-fluorescent dye that stains acidic compartments in live cells),and LysoSensor Yellow/Blue DND-160 (an acidotropic probe thataccumulates in acidic organelles due to protonation).

Specific examples of stain compounds useful for studying changes inplasma membranes include FM 1-43FX (a membrane probe analog modified tocontain an aliphatic amine), Vybrant DiI (a lipophilic membrane stain),Vybrant DiO (a lipophilic membrane stain), and Vybrant DiD (a lipophilicmembrane stain).

Specific examples of stain compounds useful for studying changes in thecytoplasm include Casein AM (a cell-permeant dye used to determine cellviability in eukaryotic cells), CellTracker Green CMFDA (a fluorescentchloromethyl derivative that exhibits green fluorescence in thecytoplasm at physiological pH), and CellTracker Red CMTPX (a fluorescentchloromethyl derivative that exhibits red fluorescence in the cytoplasmat physiological pH).

Specific examples of stain compounds useful for studying changes in theendoplasmic reticulum include ER-Tracker Blue-White DPX (a photostableprobe that is selective for the endoplasmic reticulum in live cells),SelectFX Alexa Fluor 488 (primary and secondary antibody pair), andbrefeldin A BODIPY 558/568 conjugate (a labeled reversible inhibitor ofprotein transport from the endoplasmic reticulum to the Golgiapparatus).

Specific examples of stain compounds useful for studying changes in theperoxisome include SelectFX Alexa Fluor 488 (primary and secondaryantibody pair directed against peroxisomal membrane protein 70).

Specific examples of stain compounds useful for studying changes in theGolgi include anti-golgin-97 (human) mouse IgG1 monoclonal CDF4(anti-Golgin-97 antibody), NBD C6-ceramide complexed to BSA, BODIPY FLC5-ceramide complexed to BSA, and BODIPY TR C5-ceramide complexed to BSA(fluorescent ceramides are markers for Golgi Complex in living cells).

Specific examples of stain compounds useful for studying changes in thenucleoli include SYTO RNASelect green fluorescent cell stain (a cellpermeant nucleic acid stain selective for RNA).

Specific examples of stain compounds useful for studying changes in thecytoskeleton include Alexa Fluor 488 phalloidin (a high-affinity probefor F-actin conjugated to a green-fluorescent dye), rhodamine phalloidin(a high-affinity probe for F-actin conjugated to the orange-fluorescentdye tetramethylrhodamine (TRITC)), jasplakinolide (a macrocyclicpeptide), latrunculin A (binding to monomeric G-actin in a 1:1 complex),Oregon Green 488 Taxol (paclitaxel labeled at the 7-carbon with afluorescent dye), anti-alpha tubulin mouse IgG1 (enables visualizationof microtubules with an anti-mouse IgG secondary immunoreagent).

Specific examples of stain compounds useful for studying changes inlipid rafts include Vybrant Alexa Fluor 488 (chlorea toxin subunit Blabeled with a fluorescent dye), Vybrant Alexa Fluor 555 (chlorea toxinsubunit B labeled with a fluorescent dye), and Vybrant Alexa Fluor 594(chlorea toxin subunit B labeled with a fluorescent dye).

Specific examples of stain compounds useful for studying changes incalcium levels include fluo-4 AM, fura-2 AM, indo-1 AM, and rhod-2 AM.

Specific examples of stain compounds useful for studying changes inreactive oxygen species levels include5-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester(a cell permeant indicator for reactive oxygen species that isnonfluorescent until removal of the acetate groups by intracellularesterases and oxidation),6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester(a cell permeant indicator for reactive oxygen species that isnonfluorescent until removal of the acetate groups by intracellularesterases and oxidation), dihydroethidium (hydroethidine) (cell permeantstain becomes red-fluorescent ethidium and accumulates in the nucleus),aminophenyl fluorescein, hydroxyphenyl fluorescein, BODIPY 581/591 fattyacid C11, and glutathione ethyl ester biotin amide.

Specific examples of stain compounds useful for studying changes inreactive nitrogen species levels include anti-nitrotyrosine rabbit IgG(used in conjunction with an anti-rabbit IgG secondary immunoreagent),and DAF-FM diacetate (useful for quantitating low concentrations ofnitric oxide in cells).

Specific examples of stain compounds useful for studying changes insodium levels include CoroNa Green AM (exhibits increased greenfluorescence emission upon binding sodium).

Specific examples of stain compounds useful for studying changes in pHlevels include BCECF AM, 5-chloromethyl SNARF-1 acetate, and6-chloromethyl SNARF-1 acetate.

Specific examples of stain compounds useful for studying changes in zinclevels include FluoZin-3 tetrapotassium salt (green fluorescent,suitable for detection of Zn²⁺ at 1-100 mM concentrations), andRhodZin-3 AM (orange-red fluorescent indicator useful for measuring Zn²⁺in mitochondria).

The contacting step can be performed in a variety of environments orcontainers. For example, cells in a centrifuge tube, microscope slide,or multiwell plate can be contacted. Alternatively, tissues, tissueslices, or whole organisms can be contacted.

The method can further comprise an incubation step prior to thedetection step. The incubation step can vary in length depending on theoligonucleotide experiment.

The detecting step can generally be performed by any machine or methodsuitable for detecting the signal. Examples of detecting steps includeuse of a fluorescence microscope, use of a plate reader, and use of aflow cytometer. The detecting step can be qualitative or quantitative.The detecting step can be performed at one or more discrete time points,or can be done continuously in real time.

The detecting step can comprise applying light at an absorbancewavelength to the cells, and detecting light emitted at a differentwavelength. Examples of pairs of absorbance and emission wavelengths (innm) include 346/442, 402/421, 495/519, 555/565, 578/603, 590/617,650/668, 663/690, 679/702, and 749/775.

The methods can further comprise comparing the detected signal withsignal(s) detected from control samples. For example, a control cell orcells can be treated with the same stain compound, but not with theoligonucleotide molecule.

Cellular effects may be exhibited as graded responses (as compared toall-or-none responses) in individual cells and may effect differentcells in a population differently. Even in cells which share a commonclonal origin, effects may be exhibited in certain cells in a populationbut not in others. Thus, methods disclosed herein may be used todetermine the physiological status or condition of multiple cells in apopulation and compare that status or condition of those cells to thestatus or condition of cells which have not been treated.

The one or more cells can generally be any type of cell. Examples ofcells include bacterial cells, fungal cells, insect cells, and mammaliancells. The cells can be a homogeneous or heterogeneous population. Thecells can be mixtures of multiple types of cells from the same organism,or mixtures of cells from different organisms. The cells can be“wild-type” or modified through genetic engineering, viral or pathogeninfection, randomly or specifically mutagenized, and so on.

The effects of the oligonucleotide monitored can generally be anyeffect. Effects can include cell viability, cell vitality, apoptosis,cell proliferation, signal transduction, energy charge, cell morphology,the activity of one or more enzymes, membrane potential, gene expressionefficiency, cytoskeletal integrity, and the presence or absence ofvacuoles. Effects can be measured relative to a control cell of the sametype that is not treated with the oligonucleotide molecule. Depending onthe effect measured, the signal obtained from the treated cell may behigher or lower than the signal obtained from the control cell. Thesignal obtained from the treated cell may increase or decrease overtime, depending on the effect of the oligonucleotide molecule. Effectscan include changes in pH, changes in concentration of a material (e.g.calcium or sodium), changes in shape, changes in oxidation state, and soon.

Additional embodiments involve kits useful for conductingoligonucleotide experiments. The kits can comprise one or more of theoligonucleotides of the invention, and one or more of the abovedescribed stain compounds. The kits can further comprise an instructionprotocol. The kits can comprise one or more containers for holding theoligonucleotide molecule, the stain compound, or both. The kits cancomprise one or more containers for conducting the oligonucleotideexperiments. The kits can comprise one or more buffers. The kits cancomprise one or more solvents. The kits can comprise one or morepositive or negative standards. The kits can comprise positive ornegative control samples or reference samples.

Oligonucleotide Synthesis

Oligonucleotides of the invention can be synthesized by any method knownin the art, e.g., using enzymatic synthesis and chemical synthesis. Theoligonucleotides can be synthesized in vitro (e.g., using enzymaticsynthesis and chemical synthesis) or in vivo (using recombinant DNAtechnology well known in the art).

In a preferred embodiment, chemical synthesis is used. Chemicalsynthesis of linear oligonucleotides is well known in the art and can beachieved by solution or solid phase techniques. Preferably, synthesis isby solid phase methods. Oligonucleotides can be made by any of severaldifferent synthetic procedures including the phosphoramidite, phosphitetriester, H-phosphonate, and phosphotriester methods, typically byautomated synthesis methods.

Oligonucleotide synthesis protocols are well known in the art and can befound, e.g., in U.S. Pat. No. 5,830,653; WO 98/13526; Stec et al. 1984.J. Am. Chem. Soc. 106:6077; Stec et al. 1985. J. Org. Chem. 50:3908;Stec et al. J. Chromatog. 1985. 326:263; LaPlanche et al. 1986. Nucl.Acid. Res. 1986. 14:9081; Fasman G. D., 1989. Practical Handbook ofBiochemistry and Molecular Biology. 1989. CRC Press, Boca Raton, Fla.;Lamone. 1993. Biochem. Soc. Trans. 21:1; U.S. Pat. No. 5,013,830; U.S.Pat. No. 5,214,135; U.S. Pat. No. 5,525,719; Kawasaki et al. 1993. J.Med. Chem. 36:831; WO 92/03568; U.S. Pat. No. 5,276,019; and U.S. Pat.No. 5,264,423.

The synthesis method selected can depend on the length of the desiredoligonucleotide and such choice is within the skill of the ordinaryartisan. For example, the phosphoramidite and phosphite triester methodcan produce oligonucleotides having 175 or more nucleotides while theH-phosphonate method works well for oligonucleotides of less than 100nucleotides. If modified bases are incorporated into theoligonucleotide, and particularly if modified phosphodiester linkagesare used, then the synthetic procedures are altered as needed accordingto known procedures. In this regard, Uhlmann et al. (1990, ChemicalReviews 90:543-584) provide references and outline procedures for makingoligonucleotides with modified bases and modified phosphodiesterlinkages. Other exemplary methods for making oligonucleotides are taughtin Sonveaux. 1994. “Protecting Groups in Oligonucleotide Synthesis”;Agrawal. Methods in Molecular Biology 26:1. Exemplary synthesis methodsare also taught in “Oligonucleotide Synthesis—A Practical Approach”(Gait, M. J. IRL Press at Oxford University Press. 1984). Moreover,linear oligonucleotides of defined sequence, including some sequenceswith modified nucleotides, are readily available from several commercialsources.

The oligonucleotides may be purified by polyacrylamide gelelectrophoresis, or by any of a number of chromatographic methods,including gel chromatography and high pressure liquid chromatography. Toconfirm a nucleotide sequence, oligonucleotides may be subjected to DNAsequencing by any of the known procedures, including Maxam and Gilbertsequencing, Sanger sequencing, capillary electrophoresis sequencing, thewandering spot sequencing procedure or by using selective chemicaldegradation of oligonucleotides bound to Hybond paper. Sequences ofshort oligonucleotides can also be analyzed by laser desorption massspectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am.Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass Spectrom.14:83; Grotjahn et al., 1982, Nucl. Acid Res. 10:4671). Sequencingmethods are also available for RNA oligonucleotides.

The quality of oligonucleotides synthesized can be verified by testingthe oligonucleotide by capillary electrophoresis and denaturing stronganion HPLC (SAX-HPLC) using, e.g., the method of Bergot and Egan. 1992.J. Chrom. 599:35.

Other exemplary synthesis techniques are well known in the art (see,e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual, SecondEdition (1989); DNA Cloning, Volumes I and II (D N Glover Ed. 1985);Oligonucleotide Synthesis (M J Gait Ed, 1984; Nucleic Acid Hybridisation(B D Hames and S J Higgins eds. 1984); A Practical Guide to MolecularCloning (1984); or the series, Methods in Enzymology (Academic Press,Inc.)).

Uses of Oligonucleotides

The invention also features methods of inhibiting expression of aprotein in a cell including contacting the cell with one of theabove-described oligonucleotide compositions.

The oligonucleotides of the invention can be used in a variety of invitro and in vivo situations to specifically inhibit protein expression.The instant methods and compositions are suitable for both in vitro andin vivo use.

Methods of the invention may be used for determining the function of agene in a cell or an organism or for modulating the function of a genein a cell or an organism, being capable of responding to or mediatingRNA interference. The cell is preferably a eukaryotic cell or a cellline, e.g., an animal cell such as a mammalian cell, e.g., an embryoniccell, a pluripotent stem cell, a tumor cell, e.g., a teratocarcinomacell, or a virus-infected cell. The organism is preferably a eukaryoticorganism, e.g., an animal such as a mammal, particularly a human.

The invention includes methods to inhibit expression of a target gene ina cell in vitro. For example, such methods may include introduction ofRNA into a cell in an amount sufficient to inhibit expression of thetarget gene, where the RNA is a double-stranded molecule of theinvention. By way of a further example, such an RNA molecule may have afirst strand consisting essentially of a ribonucleotide sequence thatcorresponds to a nucleotide sequence of the target gene, and a secondstrand consisting essentially of a ribonucleotide sequence that iscomplementary to the nucleotide sequence of the target gene, in whichthe first and the second strands are separate complementary strands orare joined by a loop, and they hybridize to each other to form saiddouble-stranded molecule, such that the duplex composition inhibitsexpression of the target gene. The duplex composition may includemodified nucleomonomers as discussed above.

The invention also relates to a method to inhibit expression of a targetgene in an invertebrate organism. Such methods include providing aninvertebrate organism containing a target cell that contains the targetgene, in which the target cell is susceptible to RNA interference andthe target gene is expressed in the target cell. Such methods furtherinclude contacting the invertebrate organism with an RNA composition ofthe invention. For example, the RNA may be a double-stranded moleculewith a first strand consisting essentially of a ribonucleotide sequencethat corresponds to a nucleotide sequence of the target gene and asecond strand consisting essentially of a ribonucleotide sequence thatis complementary to the nucleotide sequence of the target gene. In suchcases, the first and the second ribonucleotide sequences may be separatecomplementary strands or joined by a loop, and they hybridize to eachother to form the double-stranded molecule. Finally, such methodsinclude a step of introducing the duplex RNA composition into the targetcell to thereby inhibiting expression of the target gene.

In one embodiment, the oligonucleotides of the invention can be used toinhibit gene function in vitro in a method for identifying the functionsof genes. In this manner, the transcription of genes that areidentified, but for which no function has yet been shown, can beinhibited to thereby determine how the phenotype of a cell is changedwhen the gene is not transcribed. Such methods are useful for thevalidation of genes as targets for clinical treatment, e.g., witholigonucleotides or with other therapies.

To determine the effect of a composition of the invention, a variety ofend points can be used. In addition to the assays described previouslyherein, for example, nucleic acid probes (e.g., in the form of arrays)can be used to evaluate transcription patterns produced by cells. Probescan also be used detect peptides, proteins, or protein domains, e.g.,antibodies can be used to detect the expression of a particular protein.In yet another embodiment, the function of a protein (e.g., enzymaticactivity) can be measured. In yet another embodiment, the phenotype of acell can be evaluated to determine whether or not a target protein isexpressed. For example, the ability of a composition to affect aphenotype of a cell that is associated with cancer can be tested.

In one embodiment, one or more additional agents (e.g., activatingagents, inducing agents, proliferation enhancing agents, tumorpromoters) can be added to the cells.

In another embodiment, the compositions of the invention can be used tomonitor biochemical reactions such as, e.g., interactions of proteins,nucleic acids, small molecules, or the like, for example the efficiencyor specificity of interactions between antigens and antibodies; or ofreceptors (such as purified receptors or receptors bound to cellmembranes) and their ligands, agonists or antagonists; or of enzymes(such as proteases or kinases) and their substrates, or increases ordecreases in the amount of substrate converted to a product; as well asmany others. Such biochemical assays can be used to characterizeproperties of the probe or target, or as the basis of a screening assay.For example, to screen samples for the presence of particular proteases(e.g., proteases involved in blood clotting such as proteases Xa andVIIa), the samples can be assayed, for example using probes which arefluorogenic substrates specific for each protease of interest. If atarget protease binds to and cleaves a substrate, the substrate willfluoresce, usually as a result, e.g., of cleavage and separation betweentwo energy transfer pairs, and the signal can be detected. In anotherexample, to screen samples for the presence of a particular kinase(s)(e.g., a tyrosine kinase), samples containing one or more kinases ofinterest can be assayed, e.g., using probes are peptides which can beselectively phosphorylated by one of the kinases of interest. Usingart-recognized, routinely determinable conditions, samples can beincubated with an array of substrates, in an appropriate buffer and withthe necessary cofactors, for an empirically determined period of time.If necessary, reactions can be stopped, e.g., by washing and thephosphorylated substrates can be detected by, for example, incubatingthem with detectable reagents such as, e.g., fluorescein-labeledanti-phosphotyrosine or anti-phosphoserine antibodies and the signal canbe detected.

In another embodiment, the compositions of the invention can be used toscreen for agents which modulate a pattern of gene expression. Arrays ofoligonucleotides can be used, for example, to identify mRNA specieswhose pattern of expression from a set of genes is correlated with aparticular physiological state or developmental stage, or with a diseasecondition (“correlative” genes, RNAs, or expression patterns). By theterms “correlate” or “correlative,” it is meant that the synthesispattern of RNA is associated with the physiological condition of a cell,but not necessarily that the expression of a given RNA is responsiblefor or is causative of a particular physiological state. For example, asmall subset of mRNAs can be identified which are modulated (e.g.,upregulated or downregulated) in cells which serve as a model for aparticular disease state. This altered pattern of expression as comparedto that in a normal cell, which does not exhibit a pathologicalphenotype, can serve as a indicator of the disease state (“indicator” or“correlative” genes, RNAs, or expression patterns).

Compositions which modulate the chosen indicator expression pattern(e.g., compared to control compositions comprising, for exampleoligonucleotides which comprise a nucleotide sequence which is thereverse of the oligonucleotide, or which contains mismatch bases) canindicate that a particular target gene is a potential target fortherapeutic intervention. Moreover, such compositions may be useful astherapeutic agents to modulate expression patters of cells in an invitro expression system or in in vivo therapy. As used herein,“modulate” means to cause to increase or decrease the amount or activityof a molecule or the like which is involved in a measurable reaction. Inone embodiment, a series of cells (e.g., from a disease model) can becontacted with a series of agents (e.g., for a period of time rangingfrom about 10 minutes to about 48 hours or more) and, using routine,art-recognized methods (e.g., commercially available kits), total RNA ormRNA extracts can be made. If it is desired to amplify the amount ofRNA, standard procedures such as RT-PCR amplification can be used (see,e.g., Innis et al eds., (1996) PCR Protocols: A Guide to Methods inAmplification, Academic Press, New York). The extracts (or amplifiedproducts from them) can be allowed to contact (e.g., incubate with)probes for appropriate indicator RNAs, and those agents which areassociated with a change in the indicator expression pattern can beidentified.

Similarly, agents can be identified which modulate expression patternsassociated with particular physiological states or developmental stages.Such agents can be man-made or naturally-occurring substances, includingenvironmental factors such as substances involved in embryonicdevelopment or in regulating physiological reactions.

In one embodiment, the methods described herein can be performed in a“high throughput” manner, in which a large number of target genes (e.g.,as many as about 1000 or more, depending on the particular format used)are assayed rapidly and concurrently. Further, many assay formats (e.g.,plates or surfaces) can be processed at one time. For example, becausethe oligonucleotides of the invention do not need to be testedindividually before incorporating them into a composition, they can bereadily synthesized and large numbers of target genes can be tested atone time. For example, a large number of samples, each comprising abiological sample containing a target nucleic acid molecule (e.g., acell) and a composition of the invention can be added to separateregions of an assay format and assays can be performed on each of thesamples.

Administration of Oligonucleotide Compositions

The optimal course of administration or delivery of the oligonucleotidesmay vary depending upon the desired result and/or on the subject to betreated. As used herein “administration” refers to contacting cells witholigonucleotides and can be performed in vitro or in vivo. The dosage ofoligonucleotides may be adjusted to optimally reduce expression of aprotein translated from a target nucleic acid molecule, e.g., asmeasured by a readout of RNA stability or by a therapeutic response,without undue experimentation.

For example, expression of the protein encoded by the nucleic acidtarget can be measured to determine whether or not the dosage regimenneeds to be adjusted accordingly. In addition, an increase or decreasein RNA or protein levels in a cell or produced by a cell can be measuredusing any art recognized technique. By determining whether transcriptionhas been decreased, the effectiveness of the oligonucleotide in inducingthe cleavage of a target RNA can be determined.

Any of the above-described oligonucleotide compositions can be usedalone or in conjunction with a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” includes appropriatesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutical active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, it can be used in thetherapeutic compositions. Supplementary active ingredients can also beincorporated into the compositions.

Oligonucleotides may be incorporated into liposomes or liposomesmodified with polyethylene glycol or admixed with cationic lipids forparenteral administration. Incorporation of additional substances intothe liposome, for example, antibodies reactive against membrane proteinsfound on specific target cells, can help target the oligonucleotides tospecific cell types.

Moreover, the present invention provides for administering the subjectoligonucleotides with an osmotic pump providing continuous infusion ofsuch oligonucleotides, for example, as described in Rataiczak et al.(1992 Proc. Natl. Acad. Sci. USA 89:11823-11827). Such osmotic pumps arecommercially available, e.g., from Alzet Inc. (Palo Alto, Calif.).Topical administration and parenteral administration in a cationic lipidcarrier are preferred.

With respect to in vivo applications, the formulations of the presentinvention can be administered to a patient in a variety of forms adaptedto the chosen route of administration, e.g., parenterally, orally, orintraperitoneally. Parenteral administration, which is preferred,includes administration by the following routes: intravenous;intramuscular; interstitially; intraarterially; subcutaneous; intraocular; intrasynovial; trans epithelial, including transdermal;pulmonary via inhalation; ophthalmic; sublingual and buccal; topically,including ophthalmic; dermal; ocular; rectal; and nasal inhalation viainsufflation.

Pharmaceutical preparations for parenteral administration includeaqueous solutions of the active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, or dextran, optionally, thesuspension may also contain stabilizers. The oligonucleotides of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligonucleotides may be formulated in solidform and redissolved or suspended immediately prior to use. Lyophilizedforms are also included in the invention.

Pharmaceutical preparations for topical administration includetransdermal patches, ointments, lotions, creams, gels, drops, sprays,suppositories, liquids and powders. In addition, conventionalpharmaceutical carriers, aqueous, powder or oily bases, or thickenersmay be used in pharmaceutical preparations for topical administration.

Pharmaceutical preparations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. In addition, thickeners, flavoring agents,diluents, emulsifiers, dispersing aids, or binders may be used inpharmaceutical preparations for oral administration.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives, anddetergents. Transmucosal administration may be through nasal sprays orusing suppositories. For oral administration, the oligonucleotides areformulated into conventional oral administration forms such as capsules,tablets, and tonics. For topical administration, the oligonucleotides ofthe invention are formulated into ointments, salves, gels, or creams asknown in the art.

Drug delivery vehicles can be chosen e.g., for in vitro, for systemic,or for topical administration. These vehicles can be designed to serveas a slow release reservoir or to deliver their contents directly to thetarget cell. An advantage of using some direct delivery drug vehicles isthat multiple molecules are delivered per uptake. Such vehicles havebeen shown to increase the circulation half-life of drugs that wouldotherwise be rapidly cleared from the blood stream. Some examples ofsuch specialized drug delivery vehicles which fall into this categoryare liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres.

The described oligonucleotides may be administered systemically to asubject. Systemic absorption refers to the entry of drugs into the bloodstream followed by distribution throughout the entire body.Administration routes which lead to systemic absorption include:intravenous, subcutaneous, intraperitoneal, and intranasal. Each ofthese administration routes delivers the oligonucleotide to accessiblediseased cells. Following subcutaneous administration, the therapeuticagent drains into local lymph nodes and proceeds through the lymphaticnetwork into the circulation. The rate of entry into the circulation hasbeen shown to be a function of molecular weight or size. The use of aliposome or other drug carrier localizes the oligonucleotide at thelymph node. The oligonucleotide can be modified to diffuse into thecell, or the liposome can directly participate in the delivery of eitherthe unmodified or modified oligonucleotide into the cell.

The chosen method of delivery will result in entry into cells. Preferreddelivery methods include liposomes (10-400 nm), hydrogels,controlled-release polymers, and other pharmaceutically applicablevehicles, and microinjection or electroporation (for ex vivotreatments).

The pharmaceutical preparations of the present invention may be preparedand formulated as emulsions. Emulsions are usually heterogeneous systemsof one liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter.

The emulsions of the present invention may contain excipients such asemulsifiers, stabilizers, dyes, fats, oils, waxes, fatty acids, fattyalcohols, fatty esters, humectants, hydrophilic colloids, preservatives,and anti-oxidants may also be present in emulsions as needed. Theseexcipients may be present as a solution in either the aqueous phase,oily phase or itself as a separate phase.

Examples of naturally occurring emulsifiers that may be used in emulsionformulations of the present invention include lanolin, beeswax,phosphatides, lecithin and acacia. Finely divided solids have also beenused as good emulsifiers especially in combination with surfactants andin viscous preparations. Examples of finely divided solids that may beused as emulsifiers include polar inorganic solids, such as heavy metalhydroxides, nonswelling clays such as bentonite, attapulgite, hectorite,kaolin, montmorillonite, colloidal aluminum silicate and colloidalmagnesium aluminum silicate, pigments and nonpolar solids such as carbonor glyceryl tristearate.

Examples of preservatives that may be included in the emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Examples of antioxidants that may be included in the emulsionformulations include free radical scavengers such as tocopherols, alkylgallates, butylated hydroxyanisole, butylated hydroxytoluene, orreducing agents such as ascorbic acid and sodium metabisulfite, andantioxidant synergists such as citric acid, tartaric acid, and lecithin.

In one embodiment, the compositions of oligonucleotides are formulatedas microemulsions. A microemulsion is a system of water, oil andamphiphile which is a single optically isotropic and thermodynamicallystable liquid solution. Typically microemulsions are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a 4th component, generally an intermediatechain-length alcohol to form a transparent system.

Surfactants that may be used in the preparation of microemulsionsinclude, but are not limited to, ionic surfactants, non-ionicsurfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fattyacid esters, tetraglycerol monolaurate (ML3 10), tetraglycerolmonooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerolpentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerolmonooleate (MO750), decaglycerol sequioleate (S0750), decaglyceroldecaoleate (DA0750), alone or in combination with cosurfactants. Thecosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol,and 1-butanol, serves to increase the interfacial fluidity bypenetrating into the surfactant film and consequently creating adisordered film because of the void space generated among surfactantmolecules.

Microemulsions may, however, be prepared without the use ofcosurfactants and alcohol-free self-emulsifying microemulsion systemsare known in the art. The aqueous phase may typically be, but is notlimited to, water, an aqueous solution of the drug, glycerol, PEG300,PEG400, polyglycerols, propylene glycols, and derivatives of ethyleneglycol. The oil phase may include, but is not limited to, materials suchas Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain(C₈-C₁₂) mono, di, and tri-glycerides, polyoxyethylated glyceryl fattyacid esters, fatty alcohols, polyglycolized glycerides, saturatedpolyglycolized C₈-C₁₀ glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both oil/water and water/oil) have been proposed toenhance the oral bioavailability of drugs.

Microemulsions offer improved drug solubilization, protection of drugfrom enzymatic hydrolysis, possible enhancement of drug absorption dueto surfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11:1385; Ho et al., J. Pharm.Sci., 1996, 85:138-143). Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides from thegastrointestinal tract, as well as improve the local cellular uptake ofoligonucleotides within the gastrointestinal tract, vagina, buccalcavity and other areas of administration.

In an embodiment, the present invention employs various penetrationenhancers to affect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Evennon-lipophilic drugs may cross cell membranes if the membrane to becrossed is treated with a penetration enhancer. In addition toincreasing the diffusion of non-lipophilic drugs across cell membranes,penetration enhancers also act to enhance the permeability of lipophilicdrugs.

Five categories of penetration enhancers that may be used in the presentinvention include: surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants. Other agents may be utilizedto enhance the penetration of the administered oligonucleotides include:glycols such as ethylene glycol and propylene glycol, pyrrols such as2-15 pyrrol, azones, and terpenes such as limonene, and menthone.

The oligonucleotides, especially in lipid formulations, can also beadministered by coating a medical device, for example, a catheter, suchas an angioplasty balloon catheter, with a cationic lipid formulation.Coating may be achieved, for example, by dipping the medical device intoa lipid formulation or a mixture of a lipid formulation and a suitablesolvent, for example, an aqueous-based buffer, an aqueous solvent,ethanol, methylene chloride, chloroform and the like. An amount of theformulation will naturally adhere to the surface of the device which issubsequently administered to a patient, as appropriate. Alternatively, alyophilized mixture of a lipid formulation may be specifically bound tothe surface of the device. Such binding techniques are described, forexample, in K. Ishihara et al., Journal of Biomedical MaterialsResearch, Vol. 27, pp. 1309-1314 (1993), the disclosures of which areincorporated herein by reference in their entirety.

The useful dosage to be administered and the particular mode ofadministration will vary depending upon such factors as the cell type,or for in vivo use, the age, weight and the particular animal and regionthereof to be treated, the particular oligonucleotide and deliverymethod used, the therapeutic or diagnostic use contemplated, and theform of the formulation, for example, suspension, emulsion, micelle orliposome, as will be readily apparent to those skilled in the art.Typically, dosage is administered at lower levels and increased untilthe desired effect is achieved. When lipids are used to deliver theoligonucleotides, the amount of lipid compound that is administered canvary and generally depends upon the amount of oligonucleotide agentbeing administered. For example, the weight ratio of lipid compound tooligonucleotide agent is preferably from about 1:1 to about 15:1, with aweight ratio of about 5:1 to about 10:1 being more preferred. Generally,the amount of cationic lipid compound which is administered will varyfrom between about 0.1 milligram (mg) to about 1 gram (g). By way ofgeneral guidance, typically between about 0.1 mg and about 10 mg of theparticular oligonucleotide agent, and about 1 mg to about 100 mg of thelipid compositions, each per kilogram of patient body weight, isadministered, although higher and lower amounts can be used.

The agents of the invention are administered to subjects or contactedwith cells in a biologically compatible form suitable for pharmaceuticaladministration. By “biologically compatible form suitable foradministration” is meant that the oligonucleotide is administered in aform in which any toxic effects are outweighed by the therapeuticeffects of the oligonucleotide. In one embodiment, oligonucleotides canbe administered to subjects. Examples of subjects include mammals, e.g.,humans and other primates; cows, pigs, horses, and farming(agricultural) animals; dogs, cats, and other domesticated pets; mice,rats, and transgenic non-human animals.

Administration of an active amount of an oligonucleotide of the presentinvention is defined as an amount effective, at dosages and for periodsof time necessary to achieve the desired result. For example, an activeamount of an oligonucleotide may vary according to factors such as thetype of cell, the oligonucleotide used, and for in vivo uses the diseasestate, age, sex, and weight of the individual, and the ability of theoligonucleotide to elicit a desired response in the individual.Establishment of therapeutic levels of oligonucleotides within the cellis dependent upon the rates of uptake and efflux or degradation.Decreasing the degree of degradation prolongs the intracellularhalf-life of the oligonucleotide. Thus, chemically-modifiedoligonucleotides, e.g., with modification of the phosphate backbone, mayrequire different dosing.

The exact dosage of an oligonucleotide and number of doses administeredwill depend upon the data generated experimentally and in clinicaltrials. Several factors such as the desired effect, the deliveryvehicle, disease indication, and the route of administration, willaffect the dosage. Dosages can be readily determined by one of ordinaryskill in the art and formulated into the subject pharmaceuticalcompositions. Preferably, the duration of treatment will extend at leastthrough the course of the disease symptoms.

Dosage regima may be adjusted to provide the optimum therapeuticresponse. For example, the oligonucleotide may be repeatedlyadministered, e.g., several doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. One of ordinary skill in the art will readily beable to determine appropriate doses and schedules of administration ofthe subject oligonucleotides, whether the oligonucleotides are to beadministered to cells or to subjects.

Treatment of Diseases or Disorders

By inhibiting the expression of a gene, the oligonucleotide compositionsof the present invention can be used to treat any disease involving theexpression of a protein. Examples of diseases that can be treated byoligonucleotide compositions include: cancer, retinopathies, autoimmunediseases, inflammatory diseases (i.e., ICAM-1 related disorders,Psoriasis, Ulcerative Colitus, Crohn's disease), viral diseases (i.e.,HIV, Hepatitis C), and cardiovascular diseases.

In one embodiment, in vitro treatment of cells with oligonucleotides canbe used for ex vivo therapy of cells removed from a subject (e.g., fortreatment of leukemia or viral infection) or for treatment of cellswhich did not originate in the subject, but are to be administered tothe subject (e.g., to eliminate transplantation antigen expression oncells to be transplanted into a subject). In addition, in vitrotreatment of cells can be used in non-therapeutic settings, e.g., toevaluate gene function, to study gene regulation and protein synthesisor to evaluate improvements made to oligonucleotides designed tomodulate gene expression or protein synthesis. In vivo treatment ofcells can be useful in certain clinical settings where it is desirableto inhibit the expression of a protein. There are numerous medicalconditions for which antisense therapy is reported to be suitable (see,e.g., U.S. Pat. No. 5,830,653) as well as respiratory syncytial virusinfection (WO 95/22,553) influenza virus (WO 94/23,028), andmalignancies (WO 94/08,003). Other examples of clinical uses ofantisense sequences are reviewed, e.g., in Glaser. 1996. GeneticEngineering News 16:1. Exemplary targets for cleavage byoligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf kinase,p53, c-myb, and the bcr/abl fusion gene found in chronic myelogenousleukemia.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, and immunology, whichare within the skill of the art. Such techniques are explained fully inthe literature. See, for example, Molecular Cloning A Laboratory Manual,2nd Ed., ed. by Sambrook, J. et al. (Cold Spring Harbor Laboratory Press(1989)); Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel,F. et al. (Wiley, NY (1995)); DNA Cloning, Volumes I and II (D. N.Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984));Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. (1984)); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London(1987)); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weirand C. C. Blackwell, eds. (1986)); and Miller, J. Experiments inMolecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1972)).

Business Methods

The present invention also provides a system and method of providingcompany products to a party outside of the company, for example, asystem and method for providing a customer or a product distributor aproduct of the company such as a kit containing a double strandednucleic acid molecule which is capable of inhibiting expression of agene and/or instructions for inhibiting gene expression. FIG. 7 providesa schematic diagram of a product management system. In practice, theblocks in FIG. 7 can represent an intra-company organization, which caninclude departments in a single building or in different buildings, acomputer program or suite of programs maintained by one or morecomputers, a group of employees, a computer I/O device such as a printeror fax machine, a third party entity or company that is otherwiseunaffiliated with the company, or the like.

The product management system as shown in FIG. 7 is exemplified bycompany 100, which receives input in the form of an order from a partyoutside of the company, e.g., distributor 150 or customer 140, to orderdepartment 126, or in the form of materials and parts 130 from a partyoutside of the company; and provides output in the form of a productdelivered from shipping department 119 to distributor 150 or customer140. Company 100 system is organized to optimize receipt of orders anddelivery of a products to a party outside of the company in a costefficient manner, particularly instructions or a kit of the presentinvention, and to obtain payment for such product from the party.

With respect to methods of the present invention, the term “materialsand parts” refers to items that are used to make a device, othercomponent, or product, which generally is a device, other component, orproduct that company sells to a party outside of the company. As such,materials and parts include, for example, nucleotides, single strandedor double stranded nucleic acid molecules, host cells, enzymes (e.g.,polymerases), amino acids, culture media, buffers, paper, ink, reactionvessels, etc. In comparison, the term “devices”, “other components”, and“products” refer to items sold by the company. Devices are exemplifiedby nucleic acid molecules that are to be sold by the company, forexample, single stranded or double stranded nucleic acid molecules whichmay or may not contain one or more chemical modifications in one or bothstrands. Other components are exemplified by instructions, includinginstructions for determining a ratio of nucleic acid molecules to becombined with cells for optimal inhibition of gene expression accordingto a method of the invention. Other components also can be items thatmay be included in a kit, e.g., a kit product containing, for example,single stranded or double stranded nucleic acid molecules or cells ofone or more type (e.g., 293 cells, HUVEC cells, etc). As such, it willbe recognized that an item useful as materials and parts as definedherein further can be considered an “other component”, which can be soldby the company. The term “products” refers to devices, other components,or combinations thereof, including combinations with additionalmaterials and parts, that are sold or desired to be sold or otherwiseprovided by a company to one or more parties outside of the company.Products are exemplified herein by kits, which can contain instructionsaccording to the present invention, and single stranded or doublestranded nucleic acid molecules, or combinations thereof.

Referring to FIG. 7, company 100 includes manufacturing 110 andadministration 120. Devices 112 and other components 114 are produced inmanufacturing 110, and can be stored separately therein such as indevice storage 113 and other component storage 115, respectively, or canbe further assembled and stored in product storage 117. Materials andparts 130 can be provided to company 100 from an outside source and/ormaterials and parts 114 can be prepared in company, and used to producedevices 112 and other components 116, which, in turn, can be assembledand sold as a product. Manufacturing 110 also includes shippingdepartment 119, which, upon receiving input as to an order, can obtainproducts to be shipped from product storage 117 and forward the productto a party outside the company.

For purposes of the present invention, product storage 117 can storeinstructions, for example, for determining transfection conditions whichare suitable for use with a particular cell type or how to design adouble stranded nucleic acid molecule which will function for inhibitinggene expression, as well as combinations of such instructions and/orkits. Upon receiving input from order department 126, for example, thatcustomer 140 has ordered such a kit and instructions, shippingdepartment 119 can obtain from product storage 117 such kit forshipping, and can further obtain such instructions in a written form toinclude with the kit, and ship the kit and instructions to customer 140(and providing input to billing department 124 that the product wasshipped; or shipping department 119 can obtain from product storage 117the kit for shipping, and can further provide the instructions tocustomer 140 in an electronic form, by accessing a database in company100 that contains the instructions, and transmitting the instructions tocustomer 140 via the internet (not shown).

As further exemplified in FIG. 7, administration 120 includes orderdepartment 126, which receives input in the form of an order for aproduct from customer 140 or distributor 150. Order department 126 thenprovides output in the form of instructions to shipping department 119to fill the order, i.e., to forward products as requested to customer140 or distributor 150. Shipping department 119, in addition to fillingthe order, further provides input to billing department 124 in the formof confirmation of the products that have been shipped. Billingdepartment 124 then can provide output in the form of a bill to customer140 or distributor 150 as appropriate, and can further receive inputthat the bill has been paid, or, if no such input is received, canfurther provide output to customer 140 or distributor 150 that suchpayment may be delinquent. Additional optional component of company 100include customer service department 122, which can receive input fromcustomer 140 and can provide output in the form of feedback orinformation to customer 140. Furthermore, although not shown in FIG. 7,customer service 122 can receive input or provide output to any othercomponent of company. For example, customer service department 122 canreceive input from customer 140 indicating that an ordered product wasnot received, wherein customer service department 122 can provide outputto shipping department 119 and/or order department 126 and/or billingdepartment 124 regarding the missing product, thus providing a means toassure customer 140 satisfaction. Customer service department 122 alsocan receive input from customer 140 in the form of requested technicalinformation, for example, for confirming that instructions of theinvention can be applied to the particular need of customer 140, and canprovide output to customer 140 in the form of a response to therequested technical information.

As such, the components of company 100 are suitably configured tocommunicate with each other to facilitate the transfer of materials andparts, devices, other components, products, and information withincompany 100, and company 100 is further suitably configured to receiveinput from or provide output to an outside party. For example, aphysical path can be utilized to transfer products from product storage117 to shipping department 119 upon receiving suitable input from orderdepartment 126. Order department 126, in comparison, can be linkedelectronically with other components within company 100, for example, bya communication network such as an intranet, and can be furtherconfigured to receive input, for example, from customer 140 by atelephone network, by mail or other carrier service, or via theinternet. For electronic input and/or output, a direct electronic linksuch as a T1 line or a direct wireless connection also can beestablished, particularly within company 100 and, if desired, withdistributor 150 or materials or parts 130 provider, or the like.

Although not illustrated, company 100 system one or more data collectionsystems, including, for example, a customer data collection system,which can be realized as a personal computer, a computer network, apersonal digital assistant (PDA), an audio recording medium, a documentin which written entries are made, any suitable device capable ofreceiving data, or any combination of the foregoing. Data collectionsystems can be used to gather data associated with a customer 140 ordistributor 150, including, for example, a customer's shipping addressand billing address, as well as more specific information such as thecustomer's ordering history and payment history, such data being useful,for example, to determine that a customer has made sufficient purchasesto qualify for a discount on one or more future purchases.

Company 100 can utilize a number of software applications to providecomponents of company 100 with information or to provide a party outsideof company access to one or more components of company 100, for example,access to order department 126 or customer service department 122. Suchsoftware applications can comprise a communication network such as theInternet, a local area network, or an intranet. For example, in aninternet-based application, customer 140 can access a suitable web siteand/or a web server that cooperates with order department 126 such thatcustomer 140 can provide input in the form of an order to orderdepartment 126. In response, order department 126 can communicate withcustomer 140 to confirm that the order has been received, and canfurther communicate with shipping department 119, providing input thatproducts such as a kit of the invention, which contains, for example, adouble-stranded nucleic acid molecule and instructions for use, shouldbe shipped to customer 140. In this manner, the business of company 100can proceed in an efficient manner.

In a networked arrangement, billing department 124 and shippingdepartment 119, for example, can communicate with one another by way ofrespective computer systems. As used herein, the term “computer system”refers to general purpose computer systems such as network servers,laptop systems, desktop systems, handheld systems, personal digitalassistants, computing kiosks, and the like. Similarly, in accordancewith known techniques, distributor 150 can access a web site maintainedby company 100 after establishing an online connection to the network,particularly to order department 126, and can provide input in the formof an order. If desired, a hard copy of an order placed with orderdepartment 126 can be printed from the web browser application residentat distributor 150.

The various software modules associated with the implementation of thepresent invention can be suitably loaded into the computer systemsresident at company 100 and any party outside of company 100 as desired,or the software code can be stored on a computer-readable medium such asa floppy disk, magnetic tape, or an optical disk. In an onlineimplementation, a server and web site maintained by company 100 can beconfigured to provide software downloads to remote users such asdistributor 150, materials and parts 130, and the like. When implementedin software, the techniques of the present invention are carried out bycode segments and instructions associated with the various process tasksdescribed herein.

Accordingly, the present invention further includes methods forproviding various aspects of a product (e.g., a kit and/or instructionsof the invention), as well as information regarding various aspects ofthe invention, to parties such as the parties shown as customer 140 anddistributor 150 in FIG. 7. Thus, methods for selling devices, productsand methods of the invention to such parties are provided, as aremethods related to those sales, including customer support, billing,product inventory management within the company, etc. Examples of suchmethods are shown in FIG. 7, including, for example, wherein materialsand parts 130 can be acquired from a source outside of company 100(e.g., a supplier) and used to prepare devices (e.g., double-strandednucleic acid molecules) used in preparing a composition or practicing amethod of the invention, for example, kits, which can be maintained asan inventory in product storage 117. It should be recognized thatdevices 112 can be sold directly to a customer and/or distributor (notshown), or can be combined with one or more other components 116, andsold to a customer and/or distributor as the combined product. The othercomponents 116 can be obtained from a source outside of company 100(materials and parts 130) or can be prepared within company 100(materials and parts 114). As such, the term “product” is used generallyherein to refer an item sent to a party outside of the company (acustomer, a distributor, etc.) and includes items such as devices 112,which can be sent to a party alone or as a component of a kit or thelike.

At the appropriate time, the product is removed from product storage117, for example, by shipping department 119, and sent to a requestingparty such as customer 140 or distributor 150. Typically, such shippingoccurs in response to the party placing an order, which is thenforwarded the within the organization as exemplified in FIG. 7, andresults in the ordered product being sent to the party. Data regardingshipment of the product to the party is transmitted further within theorganization, for example, from shipping department 119 to billingdepartment 124, which, in turn, can transmit a bill to the party, eitherwith the product, or at a time after the product has been sent. Further,a bill can be sent in instances where the party has not paid for theproduct shipped within a certain period of time (e.g., within 30 days,within 45 days, within 60 days, within 90 days, within 120 days, withinfrom 30 days to 120 days, within from 45 days to 120 days, within from60 days to 120 days, within from 90 days to 120 days, within from 30days to 90 days, within from 30 days to 60 days, within from 30 days to45 days, within from 60 days to 90 days, etc.). Typically, billingdepartment 124 also is responsible for processing payment(s) made by theparty. It will be recognized that variations from the exemplified methodcan be utilized; for example, customer service department 122 canreceive an order from the party, and transmit the order to shippingdepartment 119 (not shown), thus serving the functions exemplified inFIG. 7 by order department 126 and the customer service department 122.

Methods of the invention also include providing technical service toparties using a product, particularly a kit of the invention. While sucha function can be performed by individuals involved in product researchand development, inquiries related to technical service generally arehandled, routed, and/or directed by an administrative department of theorganization (e.g., customer service department 122). Oftencommunications related to technical service (e.g., solving problemsrelated to use of the product or individual components of the product)require a two way exchange of information, as exemplified by arrowsindicating pathways of communication between customer 150 and customerservice department 122.

As mentioned above, any number of variations of the process exemplifiedin FIG. 7 are possible and within the scope of the invention.Accordingly, the invention includes methods (e.g., business methods)that involve (1) the production of products (e.g., double-strandednucleic acid molecules, transfection reagents, kits that containinstructions for performing methods of the invention, etc.); (2)receiving orders for these products; (3) sending the products to partiesplacing such orders; (4) sending bills to parties obliged to pay forproducts sent to such; and/or (5) receiving payment for products sent toparties. For example, methods are provided that comprise two or more ofthe following steps: (a) obtaining parts, materials, and/or componentsfrom a supplier; (b) preparing one or more first products (e.g., one ormore double-stranded nucleic acid molecules); (c) storing the one ormore first products of step (b); (d) combining the one or more firstproducts of step (b) with one or more other components to form one ormore second products (e.g., a kit); (e) storing the one or more firstproducts of step (b) or one or more second products of step (d); (f)obtaining an order a first product of step (b) or a second product ofstep (d); (g) shipping either the first product of step (b) or thesecond product of step (d) to the party that placed the order of step(f); (h) tracking data regarding to the amount of money owed by theparty to which the product is shipped in step (g); (i) sending a bill tothe party to which the product is shipped in step (g); (j) obtainingpayment for the product shipped in step (g) (generally, but notnecessarily, the payment is made by the party to which the product wasshipped in step (g); and (k) exchanging technical information betweenthe organization and a party in possession of a product shipped in step(d) (typically, the party to which the product was shipped in step (g)).

The present invention also provides a system and method for providinginformation as to availability of a product (e.g., a device product, akit product, and the like) to parties having potential interest in theavailability of the kit product. Such a method of the invention, whichencompasses a method of advertising to the general or a specifiedpublic, the availability of the product, particularly a productcomprising instructions and/or a kit of the present invention, can beperformed, for example, by transmitting product description data to anoutput source, for example, an advertiser; further transmitting to theoutput source instructions to publish the product information data inmedia accessible to the potential interested parties; and detectingpublication of the data in the media, thereby providing information asto availability of the product to parties having potential interest inthe availability of the product.

Accordingly, the present invention provides methods for advertisingand/or marketing devices, products, and/or methods of the invention,such methods providing the advantage of inducing and/or increasing thesales of such devices, products, and/or methods. For example,advertising and/or marketing methods of the invention include those inwhich technical specifications and/or descriptions of devices and/orproducts; methods of using the devices and/or products; and/orinstructions for practicing the methods and/or using the devices and/orproducts are presented to potential interested parties, particularlypotential purchasers of the product such as customers, distributors, andthe like. In particular embodiments, the advertising and/or marketingmethods involve presenting such information in a tangible form or in anintangible to the potential interested parties. As disclosed herein andwell known in the art, the term “intangible form” means a form thatcannot be physically handled and includes, for example, electronic media(e.g., e-mail, internet web pages, etc.), broadcasts (e.g., television,radio, etc.), and direct contacts (e.g., telephone calls betweenindividuals, between automated machines and individuals, betweenmachines, etc.); whereas the term “tangible form” means a form that canbe physically handled.

FIG. 8 provides a schematic diagram of an information providingmanagement system as encompassed within the present invention. Inpractice, the blocks in FIG. 8 can represent an intra-companyorganization, which can include departments in a single building or indifferent buildings, a computer program or suite of programs maintainedby one or more computers, a group of employees, a computer I/O devicesuch as a printer or fax machine, a third party entity or company thatis otherwise unaffiliated with the company, or the like.

The information providing management system as shown in FIG. 8 isexemplified by company 200, which makes, purchases, or otherwise makesavailable devices and methods 210 that alone, or in combination, provideproducts 220, for example, instructions, devices and/or kits of thepresent invention, that company 200 wishes to sell to interestedparties. To this end, product descriptions 230 are made, providinginformation that would lead potential users to believe that products 220can be useful to user. In order to effect transfer of productdescriptions 230 to the potential users, product descriptions 230 isprovided to advertising agency 240, which can be an entity separate fromcompany 200, or to advertising department 260, which can be an entityrelated to company 200, for example, a subsidiary. Based on the productdescriptions 230, advertisement 250 is generated and is provided tomedia accessible to potential purchasers of products 260, whom may thencontact company 200 to purchase products 220.

By way of example, product descriptions 230 can be in a tangible formsuch as written descriptions, which can be delivered (e.g., mailed,couriered, etc) to advertising agency 240 and/or advertising department250, or can be in an intangible form such as entered into and stored ina database (e.g., on a computer, in an electronic media, etc.) andtransmitted to advertising agency 240 and/or advertising department 250over a telephone line, T1 line, wireless network, or the like.Similarly, advertisement 250 can be a tangible or intangible form suchthat it conveniently and effectively can be provided to potentialparties of interest (e.g., potential purchasers of product 260). Forexample, advertisement 250 can be provided in printed form as flyers(e.g., at a meeting or other congregation of potential interestedparties) or as printed pages (or portions thereof) in magazines known tobe read by the potential interested parties (e.g., trade magazines,journals, newspapers, etc.). In addition, or alternatively,advertisement 250 can be provided in the form of directed mailing ofcomputer media containing the advertisement (e.g., CDs, DVDs, floppydiscs, etc.) or of e mail (i.e., mail or e-mail that is sent only toselected parties, for example, parties known to members of anorganization that includes or is likely to include potential users ofproducts 220); of web pages (e.g., on a website provided by company 200,or having links to the company 200 website); or of pop-up or pop-underads on web pages known to be visited by potential purchaser of products260, and the like. Potential purchasers of products 260, upon beingapprised of the availability of the products 220, for example, the kitsof the present invention, then can contact company 200 and, if sodesired, can order said products 220 for company 200 (see FIG. 7).

Kits and Instructions:

The invention also provides kits. In various aspects, a kit of theinvention may contain one or more (e.g., one, two, three, four, five,six, seven, etc.) of the following components: (1) one or more sets ofinstructions, including, for example, instructions for performingmethods of the invention or for preparing and/or using compositions ofthe invention; (2) one or more cells, including, for example, one ormore mammalian cells, for example, cells that are adapted for growth ina tissue culture medium, (3) one or more oligonucleotide or doublestranded nucleic acid molecule (including one or more control nucleicacid molecule, as described elsewhere herein); (4) one or more containercontaining water (e.g., distilled water) or other aqueous or liquidmaterial; (5) one or more containers containing one or more buffers,which can be buffers in dry, powder form or reconstituted in a liquidsuch as water, including in a concentrated form such as 2×, 3×, 4×, 5×,etc.); and/or (6) one or more containers containing one or more salts(e.g., sodium chloride, potassium chloride, magnesium chloride, whichcan be in a dry, powder form or reconstituted in a liquid such aswater).

A kit of the invention can include an instruction set, or theinstructions can be provided independently of a kit. Such instructionsmay provide information regarding how to make or use one or more of thefollowing items: (1) one or more control nucleic acid molecule (e.g., anucleic acid molecule which may be used as a transfection control); (2)one or more double stranded nucleic acid molecule, as describedelsewhere herein (e.g., a double stranded nucleic acid molecule which iscapable of “knocking-down” expression of a gene where introduced into aeukaryotic cell); (3) one or more cell lines that contain a gene theexpression of which is to be knocked down (e.g., pre-transfection growthconditions; transfection protocols; post-transfection growthconditions); (4) one or more dyes for distinguishing live from deadcells (e.g., Dead Red stain or Dead Cell Reagent), and/or (5) one ormore sets of instructions for using kit components.

Instructions can be provided in a kit, for example, written on paper orin a computer readable form provided with the kit, or can be madeaccessible to a user via the internet, for example, on the world wideweb at a URL (uniform resources link; i.e., “address”) specified by theprovider of the kit or an agent of the provider. Such instructionsdirect a user of the kit or other party of particular tasks to beperformed or of particular ways for performing a task. In one aspect,the instructions instruct a user of how to perform methods of theinvention. In a specific aspect, the instructions can, for example,instruct a user of a kit as to reaction conditions for knocking-downgene expression, including, for example, buffers, temperature, and/ortime periods of incubations for using nucleic acid molecules describedherein. Instructions of the invention can be in a tangible form, forexample, printed or otherwise imprinted on paper, or in an intangibleform, for example, present on an internet web page at a defined andaccessible URL. Thus, the invention includes instructions for performingmethods of the invention and/or for preparing compositions of theinvention. While the instructions themselves are one aspect of theinvention, the invention also includes the instructions in tangibleform. Thus, the invention includes computer media (e.g., hard disks,floppy disks, CDs, etc.) and sheets of paper (e.g., a single sheet ofpaper, a booklet, etc.) which contain the instructions.

It will be recognized that a full text of instructions for performing amethod of the invention or, where the instructions are included with akit, for using the kit, need not be provided. One example of a situationin which a kit of the invention, for example, would not contain suchfull length instructions is where the provided directions inform a userof the kits where to obtain instructions for practicing methods forwhich the kit can be used. Thus, instructions for performing methods ofthe invention can be obtained from internet web pages, separately soldor distributed manuals or other product literature, etc. The inventionthus includes kits that direct a kit user to one or more locations whereinstructions not directly packaged and/or distributed with the kits canbe found. Such instructions can be in any form including, but notlimited to, electronic or printed forms.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 Oligonucleotide Compositions Comprising ChimericAntisense Sequences

A gapped antisense oligonucleotide comprising 2′-O-methyl RNA arms andan unmodified DNA gap was synthesized. A complementary oligonucleotidewas also synthesized using unmodified RNA. A double-stranded duplex wasformed and the composition was found to inhibit expression of the targetgene.

Example 2 Length of Double-Stranded Oligonucleotides and the Presence orAbsence of Overhangs has no Effect on Function

Twenty one and 27-mers were designed to target each of two sites on thep53 molecule (89-90 site, and 93-94 site). The double-stranded moleculeswere designed with or without 3′-deoxy TT overhangs. The testoligonucleotides were 21-mers with 2 nucleotide 3′ deoxy TT overhangsand without overhangs (blunt ends); and 27-mers with 2 nucleotide 3′deoxy TT overhangs and without overhangs (blunt ends). Two positivecontrols were included in the experiment (p53) and two negative controlswere also included (FITC).

A549 cells were transfected with 100 nM of the double-stranded moleculesplus 2 ug/mL LIPOFECTAMINE™ 2000. A549 cells were examined 24 hourspost-transfection. FITC-labeled molecules were taken up well by cells.Both 21-mers (with or without overhangs) and 27-mers (with or withoutoverhangs) were non-toxic to cells. FIG. 14 shows the result of anexperiment comparing the ability of different oligonucleotide constructsto inhibit p53 and shows that length or the presence or absence of a 3′deoxy TT overhang did not affect the activity of the oligonucleotide.The results in FIG. 14 show the amount of p53 mRNA normalized to theamount of an irrelevant message, GAPDH. The level of mRNA was determinedusing RT-PCR analysis. The observed percent inhibition of p53 expressionis shown below:

21-MER 27-MER SITE overhang no overhang overhang no overhang 93-94 58%65% 62% 62% 89-90 81% 75% 67% 70%

Similar results were observed for β-3-integrin; both 21-mer and 27-merdouble-stranded molecules were found to inhibit integrin mRNA. Twodouble-stranded RNA complexes designed to target the same site of theβ-3-integrin gene were transfected in HMVEC cells. Both complexescontained a two nucleotide (TT) overhang: one complex was a 21-mer (with19 nucleotides complementary to the target gene) and the other was a27-mer (with 25 nucleotides complementary to the target gene). RT-PCRanalysis showed that the two complexes inhibited the target gene to thesame extent. HMVEC cells were transfected using 100 nM oligomercomplexed with 2.0 ug/mL of LIPOFECTAMINE™ 2000 in media containingserum for 24 hours. Twenty-four hours after transfection, the cells werelysed and the RNA was isolated for analysis by RT-PCR. No significanttoxicity was observed. The results in FIG. 14B show the amount ofβ-3-integrin mRNA normalized to the amount of GAPDH, as determined byRT-PCR analysis.

Example 3 Activation of the Double-Stranded RNA, Interferon-InducibleProtein Kinase, PKR

PKR is activated by double-stranded RNA molecules. Active PKR leads tothe inhibition of protein synthesis, activation of transcription, and avariety of other cellular effects, including signal transduction, celldifferentiation, cell growth inhibition, apoptosis, and antiviraleffects. The effect of p53-targeted double-stranded RNA molecules on PKRexpression was tested. The level of mRNA was determined using RT-PCRanalysis. As shown in FIG. 15, no correlation was observed between thelength of the double-stranded oligonucleotide and the level of PKRinduction. Accordingly, long oligonucleotides can be used withoutactivating PKR, a marker for interferon induction.

As illustrated in FIG. 15B, analysis of relative amounts of PKR mRNAafter the 21- and 27-mer transfection in HMVEC cells showedapproximately a 2 fold increase in PKR mRNA of the siRNA controlsequences over no treatment, and approximately a 2 fold increase of PKRmRNA of the 27-mer compared to the 21-mer targeted double-stranded RNAcomplexes.

Example 4 The Effect of 5′ vs. 3′ Modification on the Activity ofDouble-Stranded Oligonucleotides

Oligonucleotide duplexes were modified at either the 3′ or 5′ end withFITC groups. The modifications were made on either the antisense strandor the sense strand. 5′ or 3′ modification of the sense strand had noeffect on the percent inhibition of p53 mRNA. 3′ modification of theantisense strand had little affect on activity, while 5′ modification ofthe antisense strand reduced activity significantly. 3′ modification ofboth strands also had little affect on activity, while 3′ and 5′modification of both strands reduced activity. See FIG. 16.

The effect of the size of the group used to modify the 5′ end wastested. The results of this experiment are shown in FIG. 17. Theinclusion of a 5′ phosphate group had little effect on activity, whereasthe modification of the antisense strand or both strands had a greatereffect. The inclusion of a propyl group had more of an effect, with a 5′propyl group on the antisense strand showing a large reduction inactivity; there was also an effect when this group was added to bothstrands. Similarly, the inclusion of a FITC group at the 5′ end of theantisense molecule (or to both molecules) also significantly reduced theactivity of the RNA duplex.

Example 5 Comparison of the Efficacy of 2′-O-Methyl Modified andUnmodified Double-Stranded RNA Oligonucleotides

A549 cells were transfected with modified or unmodified RNA duplexescomplexed at 100 nM with 2 ug/mL LIPOFECTAMINE™ 2000 (Invitrogen) andwere transfected for 24 hours. The A549 cells were plated at 20,000/wellin 48 well plates. After 24 hours, FITC-labeled double-strandedoligonucleotides were visible in A549 cells; the inclusion of a2′-O-methyl group did not affect uptake. The Table below shows theresults of this experiment.

2′O-Methyl Oligonucleotide Duplexes Anti- Anti- Anti- Anti- sense/Sensesense/Sense sense/Sense sense/Sense 2′-O—Me/2′-O—Me 2′-O—Me/RNARNA/2′-O—Me RNA/RNA targeted 18639/18640 18639/16194 16193/18640 18876non- 19039/19040 19039/19044 19043/19040 18850 & targeted 16197/16198FITC-2′-O—Me/ FITC-2′-O—Me/ FITC-2′-O—Me/ 2′-O—Me/ FITC 2′-O—Me FITC-RNARNA FITC-RNA non- 19209 19037/19042 19037/19044 19039/19042 targeted

The affect of 2′-O-methyl modifications to one or both strands of adouble-stranded RNA molecule is shown in FIG. 18.

Example 6 Toxicity of p53-Targeted siRNAs in A549 Cells

27-mer siRNAs targeting p53 were not toxic to cells when compared tostandard 21-mer siRNAs having 3′ deoxy TT overhangs. In this experiment,both siRNA constructs inhibited p53 to a similar extent (83% inhibitionfor 27-mer vs. 90% inhibition for 21-mer). siRNAs were designed totarget p53 and were constructed as blunt-end 27-mers or as 21-mers with3′ deoxy TT overhangs. A549 cells were plated at 20,000 cells per wellin 48-well plates on the day prior to transfection. On the day oftransfection, cells were approximately 60-70% confluent. Cells weretransfected with 100 nM siRNAs complexed with 2 ug/mL LIPOFECTAMINE™2000 for 24 hours. Following transfection, cells were stained with DeadRed stain to visualize the extent of cell death. The siRNA sequencesused were as follows:

21-mer with overhangs targeted (5′-3′): (SEQ ID NO: 21)ACCUCAAAGCUGUUCCGUCTT (SEQ ID NO: 22) GACGGAACAGCUUUGAGGUTTBlunt-end 27-mer targeted (5′-3′): (SEQ ID NO: 23)ACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: 24) GGGACGGAACAGCTTTGAGGTGTGCGT

Example 7 Toxicity of Blunt-End 27-mer siRNAs Targeting p53 in A549Cells

The toxicity of targeted blunt-end 27-mer siRNAs targeting p53 wasobserved to be not significantly different than a control nucleic acidor no treatment. siRNAs were designed to target p53 and were constructedas blunt-end 27-mers. The corresponding control consisted ofchemistry-matched, scrambled sequences with a similar base-paircomposition. A549 cells were plated at 20,000 cells per well in 48-wellplates on the day prior to transfection. On the day of transfection,cells were approximately 60-70% confluent. Cells were transfected with100 nM siRNAs complexed with 2 ug/mL LIPOFECTAMINE™ 2000 for 24 hours.Following transfection, the cells were stained with Dead Red stain tovisualize the extent of cell death. The siRNA sequences used were asfollows:

Blunt-end 27-mer targeted (5′-3′ on top): (SEQ ID NO: 25)ACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: 26) GGGACGGAACAGCTTTGAGGTGTGCGTCorresponding control (5′-3′ on top): (SEQ ID NO: 27)CCCTGCCTTGTCGAAACTCCACACGCA (SEQ ID NO: 28) TGCGTGTGGAGTTTCGACAAGGCAGGG

Example 8 Toxicity of Blunt End 32-mer siRNAs Targeting p53 in A549Cells

Similarly, blunt-end 32-mer siRNAs targeting p53 were not observed to betoxic to cells in comparison with a control nucleic acid and notreatment, as determined by Dead Red staining. siRNAs were designed totarget p53 and were constructed as blunt-end 32-mers. The correspondingcontrol consisted of chemistry-matched, scrambled sequences with asimilar base-pair composition. A549 cells were plated at 20,000 cellsper well in 48-well plates on the day prior to transfection. On the dayof transfection, cells were approximately 60-70% confluent. Cells weretransfected with 100 nM siRNAs complexed with 2 ug/mL LIPOFECTAMINE™2000 for 24 hours. Following transfection, cells were stained with DeadRed stain to visualize the extent of cell death. The siRNA sequencesused were as follows:

Targeted blunt-end 32-mer (5′-3′ on top:) (SEQ ID NO: 29)CCCTCACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: 30)GGGACGGAACAGCTTTGAGGTGTGCGTGAGGG Corresponding control (5′-3′ on top):(SEQ ID NO: 31) CCCTGCCTTGTCGAAACTCCACACGCACTCCC (SEQ ID NO: 32)GGGAGTGCGTGTGGAGTTTCGACAAGGCAGGG

Example 9 Inhibition of p53 by 32- and 37-mer Blunt-End siRNAs

FIG. 19 depicts the results of inhibition of p53 by 32- and 37-merblunt-end siRNAs in comparison with various control experiments. siRNAswere designed to target each of two sites (93-93 site) and (89-90 site)along the coding region of p53. siRNAs were constructed as blunt-end32-mers or blunt-end 37-mers. Positive control siRNAs were 21-mers with3′ deoxy TT overhangs. Corresponding controls consisted ofchemistry-matched, scrambled sequences with a similar base-paircomposition. A549 cells were plated at 20,000 cells per well in 48-wellplates on the day prior to transfection. On the day of transfection,cells were approximately 60-70% confluent. Cells were transfected with100 nM siRNAs complexed with 2 ug/mL LIPOFECTAMINE™ 2000 for 24 hours.Following transfection, cells were lysed and poly(A) mRNA was harvestedfor RT-PCR. Inhibition of p53 expression was determined by quantitativereal-time RT-PCR (TaqMan) analysis. Expression of p53 was standardizedby quantifying GAPDH for each sample. The data in FIG. 19 representthree separate transfections analyzed in duplicate and normalized to theinternal control (GAPDH). The siRNA sequences used were as follows(depicted with the 5′-3′ strand on top):

Targeted 32-mer (89-90 site): (SEQ ID NO: 33)CCCTCACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: 34)GGGACGGAACAGCTTTGAGGTGTGCGTGAGGG 32-mer control (89-90 site):(SEQ ID NO: 35) CCCTGCCTTGTCGAAACTCCACACGCACTCCC (SEQ ID NO: 36)GGGAGTGCGTGTGGAGTTTCGACAAGGCAGGG 32-mer targeted (93-94 site):(SEQ ID NO: 37) CCCUUCUGUCUUGAACAUGAGTTTTTTATGGC (SEQ ID NO: 38)GCCATAAAAAACTCATGTTCAAGACAGAAGGG 32-mer control (93-94 site):(SEQ ID NO: 39) CGGTATTTTTTGAGTACAAGTTCTGTCTTCCC (SEQ ID NO: 40)GGGAAGACAGAACTTGTACTCAAAAAATACCG 37-mer targeted (93-94 site):(SEQ ID NO: 41) CCCTTCTGTCTTGAACATGAGTTTTTTATGGCGGGAG (SEQ ID NO: 42)CTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGG 37-mer control (93-94 site):(SEQ ID NO: 43) GAGGGCGGTATTTTTTGAGTACAAGTTCTGTCTTCCC (SEQ ID NO: 44)GGGAAGACAGAACTTGTACTCAAAAAATACCGCCCTC 21-mer targeted (89-90 site):(SEQ ID NO: 45) ACCUCAAAGCUGUUCCGUCTT (SEQ ID NO: 46)GACGGAACAGCUUUGAGGUTT 21-mer targeted (93-94 site): (SEQ ID NO: 47)CCCUUCUGUCUUGAACAUGTT (SEQ ID NO: 48) CAUGUUCAAGACAGAAGGGTT

Example 10 Enhanced Cellular Stability of Double-Stranded 2′-O-MethylRNA

In this example, the single-stranded control oligomer was transfected at800 nM. Accumulation was observed in the nucleus at 6 hours posttransfection, however by 25 hours the fluorescence of thesingle-stranded oligomer had largely dissipated, indicating the oligomerwas no longer intact (Fisher, T., T. Terhorst, et al. (1993).“Intracellular disposition and metabolism of fluorescently-labeledunmodified and modified oligonucleotides microinjected into mammaliancells.” Nucl. Acids Res. 21:3857-3865). The relative fluorescence offluorescently-labeled oligomers transfected into A549 cells was observedto fit the following pattern:

single-stranded (800 nM) double-stranded (100 nm)  6 h ++++ +++++ 25 h ++++++

The double-stranded oligomer duplex, wherein the second strand was2′-O-methyl modified RNA, was transfected at 100 nM, and was alsoclearly visible at 6 hours post transfection. However, in contrast tothe single-stranded oligomer, the double-stranded was still largelyintact in the nucleus at 24 hours, even though the concentrationtransfected was 8-fold less, thereby demonstrating that the 2′-O-methylsecond strand stabilized the oligomer in the cell.

The oligomers were all 2′-O—CH₃ with a phosphodiester backbonecontaining 6-carboxyfluorescein (6-FAM) tethered to the 5′ hydroxyl. Thesingle-stranded control oligomer was transfected at 800 nM complexedwith 4 ug/mL of LIPOFECTAMINE™ 2000, and the double-stranded complex wastransfected at 100 nM complexed with 1 ug/mL of LIPOFECTAMINE™ 2000.

Uptake of the single and double-stranded oligonucleotides was measuredby fluorescent microscopy using an inverted microscope with anexcitation wavelength of 494 nm and an emission wavelength of 519 nm.Some aliquots of cells were also stained with Dead Red, a fluorescentreagent that measures the integrity of the cell membrane and thusdistinguishes live cells from dead cells. This reagent is excited at awavelength of 528 nm and emits a red fluorescence at 617 nm. The DeadRed stain was supplied as a 1000× stock solution and was diluted to 1×with Opti-Mem. It was applied to the cells for 20 minutes in ahumidified CO₂ incubator and then removed and replaced with Opti-Mem.Any background fluorescence can be reduced by rinsing the cells withOpti-Mem and replacing with either full growth media (e.g., DMEM withsupplements) or with fresh Opti-Mem before fluorescence microscopy.

Fluorescent signal was seen accumulating in the nucleus at 6 hours posttransfection, however by 24 hours the single-stranded oligomer hassignificantly dissipated, indicating the oligomer is no longer intact.The double-stranded duplexes (wherein the second strand is 2′-O-methylmodified RNA with a 5′ 6-FAM) was transfected at 100 nM, and was alsoclearly visible at 6 hours post transfection. In contrast to thesingle-stranded oligomer, the double-stranded was still largely intactin the nucleus at 24 hours, even though the concentration transfectedwas 8-fold less. This experiment demonstrates that the 2′-O-methylsecond strand stabilizes the duplex in the cell.

Example 11 Enhanced Stability in Cells and Accumulation in Cytoplasm ofRNA Hybridized to 2′-O-Methyl RNA

The fluorescence signal, corresponding to uptake of FITC-labeled RNA and2′-O-methyl modified RNA duplexes, was measured at 6 and 24 hours. RNAcomplexes were transfected in A549 cells with 100 nM oligomer complexedwith 2 ug/mL LIPOFECTAMINE™ 2000 as described below. Cells werecontinuously transfected for 24 hours and fluorescent uptake wasassessed at 6 and 24 hours. Oligomers were 2′-O-methyl modified RNA with5′ 6-FAM (FITC-2′-O-Me), 19-mer RNA with two deoxynucleotides on the 3′end with 5′ 6-FAM (FITC-RNA) or 19-mer RNA with two deoxynucleotides onthe 3′ end (RNA) complexed. At 6 hours, the FITC-2′-O-methyl duplexesshow localization in the nucleus and the FITC-2′-O-methyl/RNA and2′-O-methyl/FITC-RNA complexes show a more diffuse pattern of uptake(these RNA/2′-O-methyl complexes are a substrate for the RISC complexand are therefore retained in the cytoplasm where the RISC complex hasbeen reported to be active). At 24 hours, the FITC-2′-O-methyl/RNA and2′-O-methyl/FITC-RNA complexes were still visible in the cell, whereastypically not even the single-stranded FITC-2′-O— was visible, even whentransfected at significantly higher concentrations, demonstrating thatthe 2′-O-methyl RNA protects the RNA strand from degradation in thecell.

RNA oligomers having a phosphodiester backbone with 2′-O-methylnucleotides were synthesized using standard phosphoramidite chemistry.Oligomers were purified by denaturing polyacrylamide gel electrophoresis(PAGE). Purity of oligomers was confirmed by (PAGE) and massspectrometry. All oligomers were greater than 90% full length, and massdata obtained was consistent with expected values. Target-specific siRNAduplexes consisted of 21-nt sense and 21-nt antisense strands withsymmetric 2-nt 3′ deoxy TT overhangs. 21-nt RNAs were chemicallysynthesized using phosphoramidite chemistry. For duplex preparation,sense- and antisense oligomers (each at 50 μM) were combined in equalvolumes in annealing buffer (30 mM HEPES pH 7.0, 100 mM potassiumacetate, and 2 mM magnesium acetate), heat-denatured at 90° C. for 1 minand annealed at 37° C. for one hour. Duplexes were stored at 80° C.until used.

A549 cells (American Type Culture Collection #CCL-185) were cultured at37° C. in Dulbecco's Modified Eagle Medium (DMEM, InvitrogenCorporation, cat. no. 11960-044) supplemented with 2 mM L-glutamine, 100units/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum(FBS). HeLa cells (American Type Culture Collection #CCL-2) werecultured at 37° C. in Minimal Essential Medium (MEM, InvitrogenCorporation, cat. no. 10370-021) supplemented with 2 mM L-glutamine, 1.5g/L sodium bicarbonate, 1.0 mM sodium pyruvate, 100 units/mL penicillin,100 μg/mL streptomycin, and 10% FBS. Cells were passaged regularly tomaintain exponential growth. On the day prior to transfection, cellswere trypsinized, counted, and seeded in 48-well plates at a density of20×10³ cells per well in 250 μL fresh media. On the day of transfectioncells were typically 60-65% confluent. Transfection of siRNA duplexesand oligomers was carried out using LIPOFECTAMINE™ 2000 (InvitrogenCorporation). Briefly, a 10× stock of LIPOFECTAMINE™ 2000 was preparedin Opti-Mem (Invitrogen Corporation) and incubated at room temperaturefor 15 minutes. An equal volume of a 10× stock of siRNA duplex oroligomers in Opti-Mem was added and complexation carried out for 15minutes at room temperature. Complexes were then diluted 5-fold in fullgrowth media. Culture media was removed from each well prior to theaddition of 250 μL complexes per well. Cells were incubated at 37° C./5%CO₂ for 6 or 24 hours prior to assessing the uptake.

The results of this experiment, as well as the experiment set forth inExample 10 demonstrate that the uptake of the double-strandedoligonucleotides can be measured through the use of a detectable labelon either strand or on both strands.

Example 12 Protocol for Transfection of NHAC Cells

NHAC cells are obtained from Clonetics (San Diego, Calif. 92123). On theday prior to transfection, approximately 4.5×10³ NHAC cells are platedin CGM (an NHAC cell culture media obtained from Clontech) in each wellof a 24-well plate. At the time of transfection, the cells arepreferably between about 70-80% confluent. A 30 μg/ml, 10× stock ofLIPOFECTAMINE™ 2000 in Opti-Mem, a serum-free medium is prepared. Thissolution is allowed to stand at room temperature for at least 15 minutesprior to use. STEALTH™ RNA molecules which are 25 nucleotides in lengthare used to transfect NHAC cells are prepared as a 3 μM, 10× stock inOpti-Mem. Equal volumes of the 10× LIPOFECTAMINE™ 2000 and 10× STEALTH™RNA oligonucleotide stocks are then added and allowed the mixture toincubate for 15 minutes to allow for complexation. This mixture is thendiluted with 4 volumes of CGM medium (Clontech) to form the final 1×(300 nM STEALTH™ RNA oligonucleotide complexed with 3 μg/mlLIPOFECTAMINE™ 2000) solution for transfection.

Prior to transfection, all media is aspirated from the NHAC cellsgrowing in the 24-well plate. 0.5 ml of the 1× lipid/STEALTH™ RNAcomplex is added to each well, being careful not to let the cells dryout during the change of media. The cells are then incubated for 16-24hours at 37° C. in a humidified CO₂ incubator. The cells are thenharvested, centrifuged to obtain a cell pellet and analyzed for proteindetermination or RNA isolation.

Example 13 Protocol for Transfection of THP-1 Cells by Electroporation

THP-1 cells are maintained in RPMI 1640 media containing 4.5 g/Lglucose, 10% FBS, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 10 mMHEPES, 1 mM sodium pyruvate, 5×10⁻⁵M 2-mercaptoethanol and Pen/Strep.The cells are split 1:3 twice a week to maintain a cell concentration of1-2×10⁶ cells/ml.

On the day prior to electroporation, THP-1 cells are seeded at0.5×10⁶/ml. The cells are collected by centrifugation on the next dayand the cell pellet washed in PB-sucrose (70 ml 0.1M sodium phosphatebuffer, 272 ml of 1 M sucrose, 1 ml of 1M MgCl₂ and 657 ml water usingsterile and endotoxin-free solutions). 15 ml of PB-sucrose is used forevery 50 ml of culture that is started with. The cells are againcentrifuged and re-washed with PB-sucrose. The cells are then countedand centrifuged again. The cell pellet is resuspended in PB-sucrose at aconcentration of 15×10⁶ cells/ml.

For electroporation, 5 μl of a 1 mM stock of STEALTH™ RNAoligonucleotide is added to a sterile tube. To this 100 μl of thefinally resuspended THP-1 cells is added and mixed gently. The cells andSTEALTH™ RNA oligonucleotide are then transferred to a sterile 0.1 cmgap electroporation cuvette and electroporated using a BioRadGENEPULSERII™ with RF module at a setting of: 100 volts, 100%modulation, 25 kHz frequency, 2 msec burst duration×10 bursts with aburst interval of 100 msec. Following electroporation, the cuvette istapped to suspend any settled cells and applied on additional burst atthe above settings. The cuvette is again taped gently immediately afterthe pulse to avoid any pH gradients and 1.5 ml of warm RPMI 164 media(described above) was added. The cells are then transferred into 2 wellsof a 24-well plate (0.7 ml/well) for recovery. The cells are incubatedat 37° C. for 24 hours, pelleted, and rinsed with Opti-Mem to remove anyexcess STEALTH™ RNA oligonucleotide. The cells can then be examined forSTEALTH™ RNA oligonucleotide uptake (if the STEALTH™ RNA oligonucleotidecomprises a detectable moiety) and can be lysed for RNA isolation orprotein determination.

Example 14 Protocol for Transfection of B6 and C3H Endothelial Cells

B6 or C3H cells are grown to confluence in either a 96- or 48-wellplate. This is typically achieved by plating B6 cells at 2×10⁴cells/well in 50 μl of DMEM media (5 ml FBS, 45 mg heparin, 6 ml of 5mg/ml ECGF, 5 ml of Antibiotic-Antimycotic (Invitrogen, Carlsbad,Calif.) and DMEM to a final volume of 500 ml) for 96 well plates or6×10⁴ cells/well in 250 μl of DMEM media for 48 well plates. C3H cellsare initially plated at 3×10⁴ cells/well in 50 μl DMEM media for 96well-plates or 6×10⁴ cells/well in 250 μl of DMEM media for 48 wellplates. Initial platings are done 2 days prior to transfection.

On the day of transfection, EPEI is diluted to a final concentration of5 μM in water to create a 20× stock. A 40 μg/ml 20× stock ofLIPOFECTAMINE™ 2000 is also made by mixing 80 μl of LIPOFECTAMINE™ 2000with 2 liters of OptiMem. A 2 μM, 10× stock of aSTEALTH™ RNAoligonucleotide is prepared in OptiMem. Each of the stock solutions isallowed to sit at room temperature for 15 minutes. 42.5 μl each of theEPEI and LIPOFECTAMINE™ 2000 stocks is then mixed with 85 μl of STEALTH™RNA oligonucleotide stock and allowed them to complex for 15 minutes atroom temperature. After complexing, the mixture is diluted by adding 680μl of Opti-Mem. 50 μl of the complexed STEALTH™ RNA oligonucleotide isthen added to each well containing either B6 or C3H cells and incubatedthe cells for 2-3 hours at 37° C. 5% CO₂. The media is then apsrated offand 50 or 250 μl (depending upon whether the cells are in 48- or 96-wellplates) of DMEM medium containing 1% FBS is added and the cells areincubated overnight.

The following day, the cells are stimulated by the addition of 2 μg/mlLPS in 1% FBS for 6 hours. Following LPS stimulation, the cells can belysed for RNA extraction and protein determination.

Example 15 Exemplary Product Literature of the Invention

The text of exemplary product literature for various embodiments ofdouble-stranded oligonucleotides of the invention are disclosed below.This product literature contains descriptions of kits of the inventionand includes instructions for use of kits of the invention, andexemplary kit components. This exemplary product literature isparticularly useful for practicing various aspects of business methodsof the invention. Suitable methods and compositions of the invention aredescribed in the product literature for the BLOCK-iT™ Fluorescent Oligo,BLOCK-iT™ Transfection Optimization Kit, and Stealth™ RNA, all of whichare available from Invitrogen Corporation, Carlsbad, Calif.

BLOCK-iT™ Fluorescent Oligo

The BLOCK-iT™ Fluorescent Oligo is a fluorescein isothiocyanate(FITC)-labeled dsRNA oligomer designed for use in RNAi analysis tofacilitate assessment and optimization of cationic lipid-mediateddelivery or electroporation of dsRNA oligonucleotides into mammaliancells. Using the BLOCK-iT™ Fluorescent Oligo in RNAi studies offers thefollowing advantages: (1) provides a good indication of the transfectionefficiency with Invitrogen's Stealth™ RNA, standard unmodified siRNA, orpurified Dicer-generated siRNA; and (2) allows strong, easyfluorescence-based indication of transfection efficiency in every RNAiexperiment. The BLOCK-iT™ Fluorescent Oligo is supplied as a 20 μM or 1mM stock of FITC-labeled double-stranded RNA (dsRNA) oligomer in 100 mMKOAc, 30 mM HEPES-KOH, pH 7.4, and 2 mM MgOAc. Upon receipt, theBLOCK-iT™ Fluorescent Oligo should be stored at −20° C., and protectedfrom light.

The BLOCK-iT™ Fluorescent Oligo is a FITC-labeled, double-stranded RNAduplex with the same length, charge, and configuration as standardsiRNA, and contains chemical modifications that enhance the stabilityand allow assessment of fluorescence signal for a significantly longertime period than is obtained with other unmodified, fluorescentlylabeled RNA. For example, fluorescence signal is readily detectable inHEK293 cells for at least 72 hours. Note that the strength of thefluorescence signal depends on the transfection efficiency, growth rateof the cells, and the amount of oligomer transfected. The sequence ofthe BLOCK-iT™ Fluorescent Oligo is not homologous to any known gene,ensuring against induction of non-specific cellular events caused byintroduction of the Oligo into cells. The Oligo also localizes primarilyto the nucleus upon uptake (Fisher et al. 1993. Nuc. Acids Res.21:3857-3865). Importantly, the BLOCK-iT™ Fluorescent Oligo is designedstrictly for use as a tool for Stealth™ RNA or siRNA uptake assessment,and is not meant to provide any information about the behavior of aStealth™ RNA or siRNA, including its cellular localization, half-life,or stability.

The BLOCK-iT™ Fluorescent Oligo is supplied as a 20 μM or 1 mM stocksolution in an annealing buffer. The guidelines below should be followedwhen handling the BLOCK-iT™ Fluorescent Oligo stock solution:

-   -   1. The BLOCK-iT™ Fluorescent Oligo is light sensitive. Store the        stock solution at −20° C., protected from light. The stock        solution is stable for at least 6 months if stored properly.    -   2. When using, thaw the stock solution on ice or at room        temperature. Once thawed, place the tube on ice until use. After        use, return stock solution to −20° C. storage.    -   3. The stock solution may be frozen and thawed multiple times        without loss of fluorescence signal if handled properly.    -   4. Take precautions to ensure that the stock solution does not        become contaminated with RNase.        -   a. Use RNase-free sterile pipette tips and supplies for all            manipulations.        -   b. Wear gloves when handling reagents and solutions.

The BLOCK-iT™ Fluorescent Oligo (20 μM or 1 mM stock) may be used withany cationic lipid-based transfection reagent suitable for delivery ofStealth™ RNA, siRNA, and Dicer-generated siRNA to mammalian cells. Forexample, Lipofectamine™ 2000 Reagent (Invitrogen Corp., Carlsbad,Calif.) provides for highly efficient transfection of a wide variety ofmammalian cells with the BLOCK-iT™ Fluorescent Oligo (Ciccarone et al.1999. Focus 21:54-55). The guidelines below should be followed whentransfecting the BLOCK-iT™ Fluorescent Oligo:

-   -   1. The amount of BLOCK-iT™ Fluorescent Oligo to use depends on        the growth rate and transfection efficiency of the mammalian        cells. When transfecting a mammalian cell line for the first        time, evaluate several concentrations of lipid and vary the        final concentration of the BLOCK-iT™ Fluorescent Oligo from 10        to 200 nM to determine the optimal amount of BLOCK-iT™        Fluorescent Oligo to use to obtain a strong fluorescence signal.        For most cell lines tested (e.g. HEK293, A549, HeLa), w a        readily detectable fluorescence signal was obtained when using        100 nM BLOCK-iT™ Fluorescent Oligo for transfection.    -   2. Prepare and seed mammalian cells at a density recommended by        the manufacturer of the transfection reagent being used.    -   3. Prepare lipid-BLOCK-iT™ Fluorescent Oligo complexes as        directed by the manufacturer of the transfection reagent being        used. Always dilute the BLOCK-iT™ Fluorescent Oligo immediately        before transfection (i.e. do not store diluted Oligo) and into        an appropriate medium. For example, the BLOCK-iT™ Fluorescent        Oligo may be diluted into Opti-MEM® I Reduced Serum Medium        (Invitrogen Corp., Carlsbad, Calif.).    -   4. Assess fluorescent uptake at 6 to 24 hours post-transfection.        The fluorescence signal may be detected at longer time points        depending on the transfection efficiency and growth rate of the        cells.

When performing electroporation, higher concentrations of BLOCK-iT™Fluorescent Oligo may be required. Use the 1 mM stock solution ofBLOCK-iT™ Fluorescent Oligo to optimize electroporation conditionsaccording to the manufacturer's guidelines.

Once the mammalian cells have been transfected or electroporated withthe BLOCK-iT™ Fluorescent Oligo, Oligo uptake in live cells may bequalitatively assessed using fluorescence microscopy. Any type offluorescence microscope and any standard FITC filter set (λ_(ex)=494 nm,λ_(em)=519 nm green) may be used for detection.

BLOCK-iT™ Transfection Optimization Kit

The BLOCK-iT™ Transfection Optimization Kit is designed to facilitateoptimization of transfection conditions for RNAi studies. The kitprovides the following reagents:

-   -   1. BLOCK-iT™ Fluorescent Oligo (20 μM FITC-labeled        double-stranded RNA (dsRNA) oligomer in annealing buffer) for        use as an indicator of transfection efficiency in RNAi        experiments with Stealth™ RNA or siRNA.    -   2. A Stealth™ RNA molecule (20 μM p53 Positive Control Stealth™        RNA in annealing buffer) targeting the human p53 gene for use as        a positive control (in human cell lines only) for the RNAi        response.    -   3. A Scrambled Stealth™ RNA molecule (20 μM Scrambled Negative        Control Stealth™ RNA in annealing buffer) for use as a negative        control (in human cell lines only) for the RNAi response.    -   4. Dead Cell Reagent (2 mM Ethidium homodimer-1 (EthD-1) in        DMSO/H₂O 1:4 (v/v)) to assess cell viability.

The annealing buffer is composed of 100 mM KOAc, 30 mM HEPES-KOH, pH7.4, and 2 mM MgOAc. Upon receipt, each of the above reagents should bestored at −20° C., and the BLOCK-iT™ Fluorescent Oligo and Dead CellReagent should be protected from light. All reagents in stock solutionsare stable for at least 6 months when stored properly.

The BLOCK-iT™ Fluorescent Oligo and the Dead Cell Reagent in theBLOCK-iT™ Transfection Optimization Kit can be used in RNAi studies tohelp optimize transfection conditions for transfecting Stealth™ RNA orsiRNA into a mammalian cell line of interest for the first time. TheBLOCK-iT™ Fluorescent Oligo allows strong, easy fluorescence-basedassessment of dsRNA oligomer uptake into mammalian cells. The BLOCK-iT™Fluorescent Oligo is functionally qualified by transient transfectioninto mammalian cells and assessment of fluorescence signal at 24 hourspost-transfection.

Dead Cell Reagent is intended for use as an indicator of cell viabilityfollowing transfection of mammalian cells with Stealth™ RNA or siRNA,and is an ethidium dye (ethidium homodimer-1; EthD-1) with the followingcharacteristics: (1) Molecular formula: C₄₆H₅₀Cl₄N₈; and (2) Molecularweight: 856.77. Dead Cell Reagent, which is excluded by the intactplasma membrane of live cells, enters cells with damaged membranes andemits a red fluorescence signal upon binding to nucleic acids(λ_(ex)=528 nm, λ_(em)=617 nm). The fluorescence signal is detectableusing a fluorescence microscope and filters for propidium iodide orTexas Red®. Once the optimal conditions to use for transfection of agiven cell line have been determined, the BLOCK-iT™ Fluorescent Oligoand the Dead Cell Reagent may be used in every RNAi experiment with thatcell line as an indicator of transfection efficiency and cell viability.

Stealth™ RNAi is chemically modified dsRNA developed to overcome thelimitations of traditional siRNA. Using Stealth™ RNA for RNAi analysisoffers the following advantages: (1) obtain effective target geneknockdown at levels that are equivalent to or greater than thoseachieved with traditional siRNA; (2) reduces non-specific effects causedby induction of cellular stress response pathways; and (3) exhibitsenhanced stability for greater flexibility in RNAi analysis. TheBLOCK-iT™ Transfection Optimization Kit includes a p53 and a ScrambledStealth™ RNA molecule for use as positive and negative controls,respectively, in an RNAi experiment targeting the human p53 gene. If themammalian cell line of interest is a human cell line that expresses p53,the p53 and Scrambled Stealth™ RNA oligomers may be used as positive andnegative controls for the RNAi response, and to help optimizetransfection conditions. The p53 and Scrambled Stealth™ RNA oligomersare functionally qualified by transient transfection into A549 cells. At24 hours post-transfection, mRNA is isolated from treated and untreatedcells using the mRNA Catcher™ Kit, and qRT-PCR is performed using LUX™primers for the human p53 gene. qRT-PCR analysis must demonstrate >75%inhibition of human p53 expression levels in p53 Stealth™ RNA-treatedcells and no inhibition in Scrambled Stealth™ RNA-treated cells.

To properly handle the reagents of the BLOCK-iT™ TransfectionOptimization Kit, the stock solutions of the BLOCK-iT™ Fluorescent Oligoand the Stealth™ RNA molecules should be thawed on ice or at roomtemperature. Once thawed, the tubes should be placed on ice until use.After use, the stock solution should be returned to −20° C. storage. Thestock solution may be frozen and thawed multiple times without loss offluorescence signal (BLOCK-iT™ Fluorescent Oligo) or activity (ControlStealth™ RNAi oligomers) if handled properly. Precautions must be takenwhen working with these reagents to ensure that the stock solutions donot become contaminated with RNase. For example, RNase-free sterilepipette tips and supplies should be used for all manipulations, andgloves worn when handling the reagents. To properly handle the Dead CellReagent, the stock solution should be thawed at room temperature. To mixthe stock solution, tap the tube, and centrifuge briefly before opening.After use, return stock solution to −20° C. storage. The stock solutionmay be frozen and thawed multiple times without loss of fluorescencesignal if handled properly.

Any suitable cationic lipid-based transfection reagent may be used todeliver Stealth™ RNA or siRNA to mammalian cells. General guidelines areprovided below for using the reagents supplied in the BLOCK-iT™Transfection Optimization Kit to help optimize transfection conditionsfor Stealth™ RNA or siRNA and mammalian cell lines. For example, theLipofectamine™ 2000 Reagent (Invitrogen Corp., Carlsbad, Calif.) isoptimal for highly efficient delivery of Stealth™ RNA or siRNA to a widevariety of mammalian cells (Gitlin et al., 2002. Nature 418:430-34; Yuet al., 2002. Proc. Natl. Acad. Sci. USA 99:6047-6052). To perform atransfection, first determine the appropriate amount of each reagent touse such that fluorescence signal (BLOCK-iT™ Fluorescent Oligo or DeadCell Reagent) or gene knockdown effect (p53 Stealth™ RNA) is readilydetectable.

The amount of BLOCK-iT™ Fluorescent Oligo used to transfect a mammaliancell line depends on the growth rate and transfection efficiency of themammalian cells. To optimize transfection conditions, evaluate severalconcentrations of lipid and vary the final concentration of theBLOCK-iT™ Fluorescent Oligo from 10 to 200 nM to determine the optimalamount of Oligo required to obtain a strong fluorescence signal. Aconcentration of 100 nM BLOCK-iT™ Fluorescent Oligo is a recommendedstarting point.

The amount of p53 Stealth™ RNA to transfect to achieve optimal geneknockdown needs to be determined experimentally for each human cellline. To optimize transfection conditions, evaluate severalconcentrations of lipid and vary the final concentration of Stealth™ RNAfrom 10 to 100 nM to determine the conditions required for the optimallevels of gene knockdown. Use of higher concentrations of Stealth™ RNAmay be possible depending on the cell line. A concentration of 40 nM p53Stealth™ RNA is a recommended starting point. The same concentration ofthe negative control Scrambled Stealth™ RNA should be used.

The transfection experiment may be set up to allow for simultaneousassessment of transfection efficiency and cell viability with the samesample by transfecting one set of cells with the BLOCK-iT™ FluorescentOligo, then staining those cells with the Dead Cell Reagent at asuitable time period after transfection (generally 6 to 24 hourspost-transfection). Prepare and seed cells at a density recommended bythe manufacturer of the transfection reagent being used. Also preparelipid-oligomer complexes as directed by the manufacturer of thetransfection reagent being used. The BLOCK-iT™ Fluorescent Oligo orStealth™ RNA should be diluted immediately before transfection (i.e. donot store diluted oligomer) and into an appropriate medium, for example,Opti-MEM® I Reduced Serum Medium (Invitrogen Corp., Carlsbad, Calif.).

The following procedure may be used to stain cells with Dead CellReagent. First, prepare a sufficient amount of the working solutionbased on the number of samples that will be stained. For example, 2mls/well of staining solution should be prepared for a 6-well culturevessel; 1 ml/well of staining solution should be prepared for a 12-wellculture vessel; 0.5 ml/well of staining solution should be prepared fora 24-well culture vessel; and 0.25 ml/well of staining solution shouldbe prepared for a 48-well culture vessel. To prepare the workingsolution, thaw the 2 mM Dead Cell Reagent stock solution at roomtemperature. Tap the tube to mix, and centrifuge briefly before opening.Dilute the appropriate amount of Dead Cell Reagent into Opti-MEM® IReduced Serum Medium to prepare a 2 μM working solution (1:1000dilution). For example, to prepare 1 ml of a 2 μM working solution, add1 μl of Dead Cell Reagent to 1 ml of Opti-MEM® I Reduced Serum Medium.Aspirate the media from the cells and replace with the appropriatevolume of Dead Cell Reagent. Incubate the cells at 37° C. in a CO₂incubator for 10-15 minutes, and then remove the Dead Cell Reagent andreplace with fresh Opti-MEM® I Reduced Serum Medium.

After the mammalian cells have been transfected with the BLOCK-iT™Fluorescent Oligo and stained with Dead Cell Reagent, Oligo uptake andcell viability may be qualitatively assessed using any fluorescencemicroscope and the following filter sets. To assess transfectionefficiency, use any standard FITC filter set (λ_(ex)=494 nm, λ_(em)=519green) to detect the fluorescence signal from the BLOCK-iT™ FluorescentOligo. To assess cell viability, use a filter set for propidium iodideor Texas Red® (λ_(ex)=528 nm, λ_(em)=617 nm) to detect the fluorescencesignal from the Dead Cell Reagent.

When the positive control Stealth™ RNA and the negative controlscrambled Stealth™

RNA are transfected into a mammalian cell line, any method of choice maybe used to detect human p53 expression levels. One exemplary method forassaying p53 mRNA levels is quantitative RT-PCR (qRT-PCR) usingInvitrogen's custom LUX™ primers. The LUX™ Designer available atwww.invitrogen.com/lux may be used to help design and order suitableprimers to use for the qRT-PCR analysis. Invitrogen's mRNA Catcher™ Kitmay be used to prepare mRNA from treated or untreated cells. Whenperforming qRT-PCR, an internal control RNA (e.g. β-actin, GAPDH, orcyclophilin) should be used to normalize results. Alternatively or inaddition, Western blot analysis using a suitable antibody to human p53may be used to assay for p53 protein levels. The half-life of theprotein should be taken into account when assessing RNAi effects at theprotein level.

Transfecting Stealth™ RNA or siRNA into Mammalian Cells UsingLipofectamine™ 2000

As described above, Lipofectamine™ 2000 Reagent is a proprietaryformulation that facilitates highly efficient delivery of Stealth™ RNAmolecules or short interfering RNA (siRNA) to mammalian cells for RNAianalysis. Below are general guidelines and a procedure to transfectStealth™ RNA or siRNA oligomers into mammalian cells usingLipofectamine™ 2000. Recommended reagent amounts are provided as astarting point. Transfection conditions for the mammalian cell line andtarget gene used should be optimized as described above for bestresults.

Many factors can influence the degree to which expression of a gene ofinterest is reduced (i.e. gene knockdown) in an RNAi experimentincluding, but not limited to, transfection efficiency, transcriptionrate of the gene of interest, protein stability, efficacy of theparticular Stealth™ RNAi or siRNA sequence chosen, and growthcharacteristics of the mammalian cell line. Take these factors intoaccount when designing transfection and RNAi experiments. For more tipsto help achieve success in RNAi experiments, refer to the section belowentitled “Seven Steps to RNAi Success.”

The following general guidelines will help optimize success whentransfecting Stealth™ RNA or siRNA into mammalian cells usingLipofectamine™ 2000. Preferably the mammalian cell line of interest usedin the transfection experiments consists of low-passage cells that arehealthy, with greater than 90% viable before transfection. The amount ofStealth™ RNA molecules or siRNA to transfect to achieve optimal geneknockdown needs to be determined experimentally for each cell line. TheStealth™ RNA molecules or siRNA of interest may be suspended inannealing buffer at a concentration of 20 μM. For example, the BLOCK-iT™Fluorescent Oligo may be used to help optimize transfection conditionsfor a cell line as described above. Once the optimal conditions to usefor transfection have been determined, the BLOCK-iT™ Fluorescent Oligomay be included in every experiment as an indicator of transfectionefficiency.

If the mammalian cell line is being transfected for the first time,evaluate several concentrations of Lipofectamine™ 2000 and vary thefinal concentration of Stealth™ RNA or siRNA from 20 to 100 nM todetermine the conditions required to achieve the optimal levels of geneknockdown. Transfecting 40 nM of Stealth™ RNA or siRNA is a goodstarting point. Higher concentrations of Stealth™ RNA or siRNA may bepossible depending on the cell line. Transfect cells at 30-50%confluence. Gene knockdown levels are generally assayed at a minimum of24 to 72 hours following transfection. Transfecting cells at a lowerdensity allows a longer interval between transfection and assay time,and minimizes the loss of cell viability due to cell overgrowth.Depending on the nature of the target gene, transfecting cells at higherdensities may be suitable with optimization of conditions. Do not addantibiotics to the medium during transfection as this reducestransfection efficiency and causes cell death. Finally, for optimalresults, use Opti-MEM® I Reduced Serum Medium (pre-warm to 37° C. beforeuse) to dilute Lipofectamine™ 2000 and Stealth™ RNA or siRNA oligomersprior to complex formation.

The following procedure may be used to transfect Stealth™ RNA or siRNAoligomers into mammalian cells using Lipofectamine™ 2000. The Tablebelow shows appropriate reagent amounts and volumes to add for differenttissue culture formats. Use the recommended amounts of Stealth™ RNA orsiRNA (see column 4) and Lipofectamine™ 2000 (see column 6) as astarting point, and optimize conditions for the cell line and Stealth™RNA or siRNA of interest (Note: 20 μM Stealth™ RNA or siRNA=20pmol/μl.).

Stealth ™ RNA Relative Volume Stealth ™ RNA or or siRNA Lipofectamine ™Lipofectamine ™ Surface of siRNA (pmol) and Amounts 2000 (μl) and 2000Amounts Culture Area (vs. Plating Dilution Volume (pmol) for DilutionVolume (μl) for Vessel 24-well) Medium (μl) Optimization (μl)Optimization 48-well 0.4 200 μl 10 pmol in 25 μl  2-25 pmol 0.5 μl in 25μl  0.3-0.8 μl 24-well 1 500 μl 20 pmol in 50 μl 10-50 pmol  1 μl in 50μl 0.5-1.5 μl  6-well 5 2 ml 100 pmol in 250 μl 50-250 pmol   5 μl in250 μl  2.5-6 μl

To begin, one day before transfection, plate cells in the appropriateamount of growth medium without antibiotics such that they will be30-50% confluent at the time of transfection. For each transfectionsample, prepare oligomer-Lipofectamine™ 2000 complexes as follows: (1)dilute Stealth™ RNA or siRNA oligomer in the appropriate amount ofOpti-MEM® I Reduced Serum Medium without serum and mix gently; (2) mixLipofectamine™ 2000 gently before use, dilute the appropriate amount inOpti-MEM® I Reduced Serum Medium, mix gently and incubate for 5 minutesat room temperature; (3) after the 5-minute incubation, combine thediluted oligomer with the diluted Lipofectamine™ 2000, mix gently andincubate for 20 minutes at room temperature to allow complex formationto occur. Note that the solution may appear cloudy, but this will notinhibit transfection. Add the oligomer-Lipofectamine™ 2000 complexes toeach well containing cells and medium. Mix gently by rocking the plateback and forth. Finally, incubate the cells at 37° C. in a CO₂ incubatorfor 24-96 hours as appropriate and then assay for gene knockdown. It isnot necessary to remove the complexes or change the medium; however,growth medium may be replaced after 4-6 hours without loss oftransfection activity.

The Table below shows optimal reagent amounts and volumes for a varietyof cell lines evaluated:

Opt. Duplex Optimal Lipid Duplex Lipid Cell Line Species TypeDescription Conc. Conc. Range Range A549 human adherent lung carcinoma100 nM 2 ug/ml L2K HeLa human adherent cervical 50 Nm 1 ug/ml L2Kadenocarcinoma MC3T3 mouse adherent fibroblast 1-100 nM 1-3 ug/mlLipofectamine 2000 (L2K) 3T3-L1 mouse adherent fibroblast 100 nM 2 ug/mlL2K (Undifferentiated) NRK rat adherent normal rat 200 nM 2 ug/ml L2Kkidney RAT 1 rat adherent fibroblast 200 nM 2 ug/ml L2K HUVEC humanadherent endothelial 300 nM 1:125 dilution of Oligofectamine HMVEC humanadherent microvascular- 200 nM 2 ug/ml L2K endothelial HEK 293 humanadherent fetal kidney 200 nM 2 u/lml L2K HepG2 human adherent hepatocyte300 nM 2 ug/ml L2K (while plating) MSC human adherent Mesenchymal 400 nM2 ug/ml L2K 100-400 nM stem cells SK-N-SH human adherent neuroblastoma300 nM 3 ug/ml L2K Keratinocytes human adherent primary 100 nM 2 ug/mlL2K 10-200 nM 2-3 ug/ml L2K keratinocytes Sebocytes human adherentprimary 100 Nm 3 ug/ml L2K sebocytes Melanocytes human adherent primary0.25-1 ug 1-3 u/l Mirus melanocytes duplex/well TKO/ug duplex (Mirus,Madison, WI, Cat No. MIR 2154) HCT 116 human adherent colorectal 400 nM4 ug/ml   200-800 nM 2-4 ug/ml carcinoma HNAC human adherent cartilage300 nM 3 ug/ml L2K C2C12 mouse adherent myoblast 50 nM 2 ug/ml L2KPrimary mouse adherent endothelial 200 nM 2 ug/ml L2K endothelium 0.25uM EPEI MCF7 human adherent breast 500 nM 3 ug/ml L2K adenocarcinoma(while plating) RAW mouse adherent osteoclast 150 nM 2 ug/ml L2K 75-300nM 264.7 Jurkat human suspension acute t-cell 50 uM. Electroporationleukemia 100 volts 50% modulation 25 Khz 2 msec 10 bursts THP-1 humansuspension acute monocytic 50 uM Electroporation leukemia 100 volts 100%modulation 25 Khz 2 msec 10 bursts Hut-78 human suspension human t-cell50 uM Electroporation 100 volts 80% n modulation 25 Khz 2 msec 10 bursts

Seven Steps Toward RNAi Success

1. Optimize Transfection Conditions Before Beginning Experiments withStealth™ RNA.

The level of confluence and passage number of the cells at the start oftransfection can have a significant impact on the efficiency of Stealth™RNA uptake and on the cellular toxicity associated with transfection.Before beginning RNAi analysis, optimize transfection conditions bydetermining the optimal cell density and oligomer-lipid concentrationsto use for the mammalian cell line of interest and system. Whenoptimizing transfection conditions, follow these guidelines: (1) Toensure uniform uptake of Stealth™ RNA, make sure that cells are plateduniformly across the wells; (2) For highly efficient transfection in abroad range of mammalian cell types, use Lipofectamine™ 2000 Reagent;(3) Use the BLOCK-iT™ Fluorescent Oligo to optimize transfectionconditions as described above. Uptake of the BLOCK-iT™ Fluorescent Oligocorrelates strongly with uptake of Stealth™ RNA or siRNA oligomers.

2. Include the BLOCK-iT™ Fluorescent Oligo in Every Experiment.

The degree of the RNAi response to a particular Stealth™ RNA or siRNAoligomer is directly linked to its transfection efficiency. To assesstransfection efficiency, include the BLOCK-iT™ Fluorescent Oligo inevery experiment. Using the BLOCK-iT™ Fluorescent Oligo in transfectionexperiments allows for easy assessment of oligomer uptake andtransfection efficiency using any fluorescence microscope and a standardFITC filter set. Uptake of the fluorescent oligomer by at least 80% ofcells correlates with high levels of gene knockdown by effectiveStealth™ RNA or siRNA oligomers. Note that the BLOCK-iT™ FluorescentOligo is chemically modified to enhance its stability and allowsassessment of fluorescence signal for a significantly longer time periodthan is obtained with other unmodified, fluorescently-labeled RNA.

3. Assess Stealth™ RNAi or siRNA Effects by Performing an RNA Assay(i.e. qRT-PCR) First.

To validate Stealth™ RNA or siRNA oligomers, measure each oligomer'seffect on the target mRNA. Although many investigators wish to bypassthe RNA determination step and look directly at the Stealth™ RNA orsiRNA oligomer's effect on protein levels, this is unadvisable, sincethe RNA assay will yield important information about the rank orderpotency of the oligomers against the target mRNA and provides valuableinformation required to troubleshoot the assay system. For example, anRNAi oligomer may be effective at decreasing mRNA levels of the targetgene; however, may not affect protein levels if the target protein has along half-life. Quantitative RT-PCR (qRT-PCR) using custom LUX™ primers(Invitrogen Corp., Carlsbad, Calif.) provides a convenient and highthroughput method to evaluate the effect of an individual or set ofStealth™ RNA or siRNA oligomers on target mRNA levels. The LUX™ Designeravailable at www.invitrogen.com/lux may be used to help design and ordersuitable primers for use in qRT-PCR analysis. To prepare mRNA or totalRNA from untreated or oligomer-treated cells, mRNA Catcher™ Kit(Invitrogen Corp., Carlsbad, Calif.) or Concert™ 96 RNA PurificationSystem (Invitrogen Corp., Carlsbad, Calif.) may be used, respectively.When performing qRT-PCR, results should be normalized to an internalcontrol RNA (e.g. β-actin or GAPDH).

4. Know the Half-Life of the Protein that you Wish to Inhibit.

To see Stealth™ RNA or siRNA-mediated inhibition at the protein level,any pre-existing pool of the protein must be degraded. If the protein ofinterest has a long half-life, long-term transfection experiments mayneed to performed (i.e. perform multiple cycles of transfection) toobserve effects at the protein level.

5. Always Include the Appropriate Positive and Negative Controls.

When performing RNAi analysis, it is important to include the properpositive and negative controls to help evaluate experimental results.For a positive control, include an effective Stealth™ RNA for a targetother than the mRNA of interest. For a negative control, compare thelevels of the target mRNA in Stealth™ RNA or siRNA-treated and control(scrambled or reverse sequences)-treated cells, for example by using theBLOCK-iT™ Transfection Optimization Kit as described previously.

6. Follow these General Guidelines to Perform RNAi Analysis usingStealth™ RNA or siRNA.

When preparing oligomer-lipid complexes, dilute oligomer and lipid intothe appropriate medium, for example Opti-MEM® I Reduced Serum Medium. Donot use phosphate-buffered saline (PBS) for dilution, as transfectionefficiency will be severely compromised. Always mix the Stealth™ RNA orsiRNA oligomer stock solution thoroughly to before use. Thaw, vortex,and spin to collect fluid before removing sample. Do not allow the cellsto dry out before adding oligomer-lipid complexes. Doing so will reducethe transfection efficiency and cell viability. For detailed protocolsto transfect Stealth™ RNA or siRNA oligomers, refer to themanufacturer's instructions for the transfection reagent being using.

7. Visit www.invitrogen.com/rnai for Additional Information, Resources,and Protocols to Help Achieve RNAi Analysis Success.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. The entire contentsof all patents, published patent applications and other references citedherein are hereby expressly incorporated herein in their entireties byreference.

1. A method for introducing a double-stranded nucleic acid moleculecomprising a first strand and a second strand into a eukaryotic cell invitro, the method comprising contacting the eukaryotic cell with thedouble-stranded nucleic acid molecule, wherein from one to six of thenucleotides at the 5′ terminus of the first strand of thedouble-stranded nucleic acid molecule are chemically modified at the 2′positions, wherein said modification is a 2′-O-methyl modification;wherein from one to six of the nucleotides at the 5′ terminus of thesecond strand of the double-stranded nucleic acid molecule arechemically modified at the 2′ positions, wherein said modification is a2′-O-methyl modification; wherein the double-stranded nucleic acidmolecule is between 18 and 30 nucleosides in length; and wherein thedouble-stranded nucleic acid molecule is introduced into the eukaryoticcell and participates in RNA interference mediated degradation of RNAwhich shares sequence complementarity with at least one strand of thedouble-stranded nucleic acid molecule.
 2. The method of claim 1, whereinthe double-stranded nucleic acid molecule is between 20 and 30nucleosides in length.
 3. The method of claim 1, wherein thedouble-stranded nucleic acid molecule is 25 nucleosides in length. 4.The method of claim 1, wherein the double-stranded nucleic acid moleculecontains an overhang of at least one nucleoside on at least one end. 5.The method of claim 4, wherein the overhang is a 3′ end overhang.
 6. Themethod of claim 4, wherein the nucleosides of the 3′ end overhang aredeoxy T-deoxy T.
 7. The method of claim 1, wherein the 2′ chemicalmodification is on a ribose.
 8. The method of claim 1, wherein thedouble-stranded nucleic acid molecule is RNA.
 9. The method of claim 1,wherein the eukaryotic cell is contacted with the double-strandednucleic molecule in the presence of a transfection reagent.
 10. Themethod of claim 9, wherein the transfection reagent is a cationic lipid.11. The method of claim 1, wherein the double-stranded nucleic acidmolecule is introduced into the eukaryotic cell by electroporation. 12.The method of claim 1, wherein a strand of said double-stranded nucleicacid molecule is complementary to a sequence of an mRNA expressed insaid eukaryotic cell.
 13. The method of claim 12, wherein saiddouble-stranded nucleic molecule is a double-stranded RNA molecule.